Activation of human natural killer cells by lipopolysaccharide and generation of interleukin-1 alpha, beta, tumour necrosis factor and interleukin-6. Effect of IL-1 receptor antagonist.

The presence in the body of an antigen species or a bacterial lipopolysaccharide (LPS) has a pleiotropic effect on the immune system activating macrophages, lymphocytes and natural killer (NK) cells. Recently it has been reported that human macrophages not only secrete interleukin-1 (IL-1) but also its inhibitor, called IL-1 receptor antagonist (IL-1ra), structurally similar to IL-1 beta, but with no IL-1-like activity and which binds to the IL-1 receptor. In this study we show that LPS stimulates NK cell activity and IL-1ra potentiates the stimulatory effect of human recombinant interleukin-2 (hrIL-2) on NK cell activity. In addition, we found that hrIL-1ra inhibits DNA synthesis in lymphocyte culture stimulated with phytohaemagglutinin (PHA) (20 micrograms/ml), presumably via IL-1 inhibition. We also found that LPS is a potent stimulator of monokines: IL-6, tumour necrosis factor-alpha (TNF-alpha), and IL-1 beta, as determined by radioimmunoassay method, and interferon-gamma (IFN-gamma), IL-2, TNF-alpha and IL-1 alpha, as determined by ELISA method, in peripheral blood mononuclear cells (PBMC). We used PBMC as effector cells since LPS requires the presence of accessory cells to activate lymphocytes and bind to the HLA-DR molecule on accessory cells. The effect of LPS on PBMC cytotoxicity has been compared with an endotoxin-free extract of Escherichia coli, OM-8990, which did not provoke cytokine production nor did it cause enhancement of NK cell activity. We found that human recombinant IL-1ra potentiates the stimulatory effect of IL-2 on NK cell activity, similar to hrIL-1 beta. The potentiation of IL-2 in stimulating NK cell activity by IL-1ra is not yet understood. Since IL-1ra is a part of the IL-1 family, it may work in a similar fashion to IL-1, which also potentiates IL-2 to enhance NK cell activity but has been shown not to be directly important in tumour cell killing. In addition, hrIL-1ra can amplify the effect of IL-2 on NK activity, possibly by inhibiting the cyclo-oxygenase products, which are immunosuppressive and are generated in antigen-stimulated PBMC cultures. The generation of IFN-gamma by PBMC after treatment with LPS strongly suggests that the enhancement of NK cell activity may be indirectly due to IFN production.     

Requirement for cell-to-cell contact for the immunosuppressive activity of human alveolar macrophages

The ability of alveolar macrophages (AM) obtained by bronchoalveolar lavage of healthy volunteers to suppress T lymphocyte responses to the mitogen phytohemagglutinin (PHA) in vitro was investigated. AM but not monocytes (MN) inhibited responses of peripheral blood mononuclear cells (PBMC) to PHA as measured by incorporation of [3H]thymidine [( 3H]TdR) and interleukin-2 (IL-2) expression. Supernatants of AM generated for various periods and with various concentrations of cells did not, however, inhibit PBMC responses to PHA. To examine the role of cell contact in the inhibitory activity of AM, AM or MN were added to PBMC in 6-well plates either directly (in co-culture) or separated by a 0.45-micron filter. MN did not inhibit PBMC blastogenic responses under either condition. AM at a 1:2 ratio with PBMC inhibited blastogenesis by 75 +/- 11% (mean +/- SD, n = 3, P less than 0.01) when cultured directly with PBMC but had no inhibitory effect on blastogenesis when physically separated from target PBMC. AM in co-culture with PBMC also inhibited PHA-stimulated IL-2 production by 70% but did not inhibit IL-2 production when AM were separated from PBMC in dual chambers. To assess the role of the cell surface in the inhibitory activity of AM, AM and MN were fixed with 2% paraformaldehyde. Neither fixed nor unfixed MN inhibited PBMC blastogenic responses, but both fixed and unfixed AM inhibited responses similarly (77 to 95%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of inhibition of mononuclear cell activation by the iron-chelating agent desferrioxamine.

Iron-withholding by the chelating agent desferrioxamine abrogates the proliferative response of human peripheral blood mononuclear cells (PBMC) to phytohaemagglutinin (PHA). The present study investigated whether desferrioxamine operates late in the activation process or, as recently suggested, at an early stage, by inhibiting the appearance of the interleukin-2 (IL-2) receptor. Human PBMC were stimulated with PHA (10 micrograms/ml) and [3H]thymidine ([3H]TdR) incorporation determined after 66 hr of culture. Greater than 90% inhibition was achieved by concentrations of desferrioxamine as low as 5 mumol/l present throughout culture, while IL-2 receptor expression (anti-Tac), analysed by FACS, was maintained at up to 75% of control levels. 300 mumol/l desferrioxamine present throughout culture abrogated [3H]TdR incorporation and additionally suppressed IL-2 receptor to 10-15% of control levels. In contrast, the same high dose of desferrioxamine when added for 2 hr to cells previously cultured for 66 hr produced 80% inhibition of [3H]TdR incorporation but failed to inhibit expression of the IL-2 receptor. Desferrioxamine rapidly achieved equilibrium across the cell membrane (within 60 min) and chelated 59Fe delivered to activated cells by the transferrin endocytic cycle. These results indicate that desferrioxamine can inhibit T-cell activation either early or late in the process by chelating iron and independently of an effect on the IL-2 receptor. In support of a dual effect of the drug is the finding that at 50 mumol/l, desferrioxamine-enhanced expression of the transferrin receptor occurred, an adaptive response made to intracellular iron depletion, while IL-2 receptor expression was inhibited.     

Modulation in vitro of human natural cytotoxicity, lymphocyte proliferative response to mitogens and cytokine production by essential fatty acids.

Essential fatty acids (EFA) have been shown in animal studies to have a differential effect on various aspects of immune reactivity. However, there have been few studies in humans. Therefore, we elected to investigate the effects of a variety of EFA [gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] in vitro on human blood lymphocyte reactivity, cytokine secretion and natural cytotoxicity. The proliferative response to polyclonal mitogens (phytohaemagglutinin, pokeweed mitogen, concanavalin A), as measured by [3H]thymidine incorporation into newly synthesized lymphocytes, was inhibited (P < 0.05) by all EFAs tested, in a dose-dependent manner (3-15 micrograms/ml). The greatest inhibition of proliferation was caused by EPA and DHA. Similarly, EPA, DHA and GLA significantly reduced cytotoxic activity [expressed as lytic units, using 51 chromium-release assays natural killer (NK) (K562 cells) and lymphokine-activated (LAK) (Daudi cells) cells] (P < 0.05) in a concentration-dependent manner (5-50 micrograms/ml), without affecting cell viability. EPA and DHA exhibited greater suppression than GLA. Furthermore, the inhibition of cell proliferation and suppression of natural cytotoxicity was associated with marked decrease in cytokine [interleukin-1 (IL-1), IL-2, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)] production in vitro. Our findings demonstrate that EFAs (GLA, EPA, DHA) have the potential to inhibit significantly various aspects of human lymphocyte cell-mediated and humoral immune reactivities.     

Suppression of T-lymphocyte blastogenesis by ovine trophoblast protein-1 and human interferon-alpha may be independent of interleukin-2 production

Cells derived from the trophoblast tissue of a day 15 sheep conceptus released substances that inhibit incorporation of [3H]thymidine into phytohemagglutinin (PHA)-stimulated ovine lymphocytes. This effect was partially reversed by addition of antiserum to ovine trophoblast protein-1 (oTP-1), a major secretory product of day 13-21 sheep conceptuses and a protein structurally and functionally related to alpha-interferons (IFN-alpha). Human IFN-alpha, unlike dexamethasone, inhibits phytohemagglutinin-induced lymphocyte blastogenesis without reducing interleukin-2 (IL-2) production by the cultures, and conditioned medium containing IL-2 does not promote [3H]thymidine incorporation into ovine lymphocytes when oTP-1 is present. Thus, oTP-1, by virtue of being an IFN, may have a local immunomodulatory role by selectively inhibiting the proliferative responses of certain maternal immune cells to IL-2.     

Activation and proliferation of normal resting human T lymphocytes in serum-free culture: role of IL-4 and IL-6

Purified human T lymphocytes, completely depleted of accessory cells [i.e. monocytes, large granular lymphocytes (LGL) and B lymphocytes], have been grown in serum-free culture in presence of a mitogenic lectin (phytohaemagglutinin, PHA) and different recombinant cytokines. Only IL-2 and IL-4 induced a marked stimulation of [3H] thymidine ([3H]TdR) uptake, cell proliferation and expression of activation markers [transferrin receptor (TrfR), IL-2R]. The other cytokines (IL-1 alpha, IL-1 beta, IFN-gamma, GM-CSF, TNF-alpha) had no significant effect, except for a moderate, but significant, stimulation of [3H]TdR uptake induced by IL-3. Simultaneous addition of IL-4 and anti-IL-2 neutralizing monoclonal antibodies (mAb) did not modify the effects induced by IL-4 alone. Furthermore, IL-2 was not detected in the supernatant of T cells grown in the presence of PHA and IL-4. Thus, our results indicate that IL-4 acts on T lymphocytes independently of IL-2. We also observed that IL-6 moderately activates DNA synthesis in PHA-stimulated T lymphocytes, but markedly potentiates the proliferative effect of suboptimal amounts of IL-2. In conclusion, the present study suggests that B-cell growth factors, in addition to IL-2, control the proliferation of normal circulating T lymphocytes.     

Inhibition of leukotriene B4(LTB4) by recombinant interleukin-1 receptor antagonist (IL-1RA) on human monocytes

Macrophages are a primary source of interleukin-1 (IL-1), a glycoprotein which plays an important and essential role in the immune response and inflammation. Cytokines stimulate many different cells to produce increasing amounts of arachidonic acid metabolites such as prostaglandins and leukotrienes. Recently, interleukin-1 receptor antagonist (IL-1ra), a natural inhibitor of IL-1 released by macrophages, has been reported to inhibit PGE₂. In accordance with these data our results show that the pretreatment, for 60 min, of purified human peripheral monocytes with IL-1ra at different concentrations (0.25–250 ng/ml) inhibits, in a dose-dependent manner, the generation of LTB₄ released after 10 min treatment with calcium ionophore A23187 (5 μM). The inhibition of LTB₄ synthesis by hrIL-1ra suggests the possibility that this new glycoprotein plays a modulatory role in immunity and inflammation.

     

Inhibition of leukotriene B4(LTB4) by recombinant interleukin-1 receptor antagonist (IL-1RA) on human monocytes

Macrophages are a primary source of interleukin-1 (IL-1), a glycoprotein which plays an important and essential role in the immune response and inflammation. Cytokines stimulate many different cells to produce increasing amounts of arachidonic acid metabolites such as prostaglandins and leukotrienes. Recently, interleukin-1 receptor antagonist (IL-1ra), a natural inhibitor of IL-1 released by macrophages, has been reported to inhibit PGE₂. In accordance with these data our results show that the pretreatment, for 60 min, of purified human peripheral monocytes with IL-1ra at different concentrations (0.25–250 ng/ml) inhibits, in a dose-dependent manner, the generation of LTB₄ released after 10 min treatment with calcium ionophore A23187 (5 M). The inhibition of LTB₄ synthesis by hrIL-1ra suggests the possibility that this new glycoprotein plays a modulatory role in immunity and inflammation.     

The use of [125I]deoxyuridine for semi-microevaluation of human lymphocyte response to phytohemagglutinin in vitro

[125I]5-iodo-2'-deoxyuridine ([125I]UdR) was compared with [6-3H]thymidine ([3H]TdR) in a semi-micromethod used to evaluate the response of human lymphocytes to phytohemagglutinin (PHA) in vitro. 5-Fluoro-2'-dexoyuridine (FUdR) was added to [125I]UdR in order to increase its incorporation, by preventing endogenous thymidine synthesis. The relationship between PHA concentration and the isotope uptake by lymphocytes was studied. Similar dose-response curves were found for the two precursors, with a peak at 15 microgram/ml of PHA. Nevertheless the recovery of the labeled compounds was higher when [125I]UdR together with FUdR was used.     

Characterization of the immunoregulatory properties of thymosin alpha 1 on interleukin-2 production and interleukin-2 receptor expression in normal human lymphocytes

Thymosin alpha 1 (T alpha 1) and thymosin fraction 5 (TF5) have been shown to induce lymphocyte maturation and differentiation as well as to modulate mature immune responses to antigens and mitogens. The present study focused on the characterization of the mechanisms involved in T alpha 1 and TF5 enhancement of phytohemagglutinin (PHA)-induced interleukin-2 (IL-2) secretion and interleukin-2 receptor (IL-2R) expression in human mononuclear cells. We provide evidence that TF5 and T alpha 1 modulate an early event(s) during lymphocyte activation by mitogens. A short preincubation period (30 min) of non-adherent cells with thymosins, followed by extensive washing and subsequent exposure to PHA, was sufficient to enhance the production of IL-2 and the expression of IL-2R induced by the mitogen. Furthermore, the concomitant addition of PHA and thymosin during the preincubation period is not necessary for the enhancing effects to occur. We have also studied the role of macrophages on thymosin modulation of these responses. Results presented here indicate that macrophages are not essential for the interaction of thymosins with T-cells. However, macrophages are an absolute requirement during the exposure to the mitogen after preincubation with thymosins for the manifestation of TF5- and T alpha 1-mediated enhancing effects on IL-2 production and IL-2R expression. Human recombinant interleukin-1 beta (rIL-1 beta) was able to replace this macrophage requirement, indicating that production of IL-1 by these cells is a critical event in thymosin modulation of the IL-2 system. Two-color flow cytometric analysis and experiments involving the use of highly purified helper/inducer (Th, CD4+) and cytotoxic/suppressor (Tc, CD8+) T-cell populations indicated that both, Th and Tc cell populations are targets of thymosin activity. These studies provide additional evidence that thymosins play an important role in the modulation of the normal immune response and begin to define the mechanisms underlying T alpha 1 immunoregulatory properties.     

Lymphocyte stimulation in vitro by orosomucoid glycoprotein

Mitogenesis of human peripheral blood lymphocytes as measured by the uptake of [3H]thymidine was stimulated in vitro by pure orosomucoid glycoprotein when used at concentrations that are considerably lower than the physiological plasma level. The lymphocyte cultures stimulated with PHA or PWM were not affected by low concentration (67 micrograms/ml), but they were mildly suppressed by high concentration (1 mg/ml) of this glycoprotein. The stimulatory response was relatively greater with fractionated T cells than the non-T cells (B cells and monocytes). At 50 micrograms/ml concentration of orosomucoid, the lymphocyte activation was found in randomly selected blood donors which included normal healthy volunteers and patients with T cell immunodeficiency or Alzheimer's disease, demonstrating a consistent immunostimulatory action of this glycoprotein.     

Inhibitory and stimulatory effects of Pseudomonas aeruginosa pyocyanine on human T and B lymphocytes and human monocytes.

Pyocyanine, a pigment produced by Pseudomonas aeruginosa, has dual dose-dependent stimulatory as well as inhibitory effects on immune responses in vitro as measured by DNA synthesis of human T and B lymphocytes, interleukin-2 (IL-2) production by human T lymphocytes, immunoglobulin production by human B lymphocytes, and monokine production by human monocytes. In general, stimulatory activity was found at low concentrations of pyocyanine, whereas high concentrations of the pigment resulted in an inhibition of responses. At a pyocyanine concentration of 0.1 micrograms/ml or less the proliferation of T and B lymphocytes was enhanced, but at 0.5 micrograms/ml it was suppressed. IL-2 production by T lymphocytes was enhanced at concentrations up to 0.5 micrograms/ml but totally inhibited at 1.0 micrograms/ml. The differentiation of B lymphocytes to become immunoglobulin-producing cells was also enhanced in the presence of low doses of pyocyanine, whereas secretion of immunoglobulin by B lymphocytes was suppressed at all concentrations of pyocyanine. In contrast to the dual effects of pyocyanine on lymphocyte response, lipopolysaccharide-induced IL-1 and tumor necrosis factor release by monocytes was markedly enhanced by low as well as high concentrations of pyocyanine. From these results we conclude that this property of pyocyanine may lead to suppression of specific defense mechanisms and enhance harmful inflammatory reactions of the host during infection with Pseudomonas aeruginosa.     

Suppression of in vitro lymphocyte DNA synthesis by killed Pseudomonas aeruginosa.

Whole antibiotic-killed classic Pseudomonas aeruginosa organisms elicited human lymphocyte [3H]thymidine (TdR) uptake in vitro after 5 days in culture. However, high concentrations of the same preparation did not elicit [3H]TdR incorporation. The investigation of this lymphocyte unresponsiveness revealed that a high dose of P. aeruginosa, when added to lymphocyte cultures together with optimal concentrations of lymphocyte activators (e.g., plant lectins or whole killed Staphylococcus aureus Cowan 1), caused a potent, nonspecifically expressed inhibition of lymphocyte [3H]TdR uptake in response to these mitogens. High doses of P. aeruginosa were not cytotoxic to lymphocytes, and the inhibition caused was reversed when lymphocytes were washed free of bacteria. The inhibition of [3H]TdR uptake by high-dose P. aeruginosa did not require the generation of adherent suppressor cells or prostaglandin-mediated, steroid-sensitive or radiation-sensitive suppressor mechanisms. At optimal lymphocyte stimulatory concentrations of P. aeruginosa, the addition of indomethacin or the depletion of adherent cells caused an increase in lymphocyte [3H]TdR incorporation. This is consistent with an adherent-cell population regulating [3H]TdR uptake in response to P. aeruginosa via a prostaglandin-dependent pathway. This population was not involved in the inhibition of lymphocyte [3H]TdR uptake by high concentrations of P. aeruginosa.     

Inhibition of lymphocyte proliferation by ovine trophoblast protein-1 and a high molecular weight glycoprotein produced by the peri-implantation sheep conceptus

Sheep conceptuses were flushed from uteri on day 16 of pregnancy and cultured in vitro. Three peaks of immunosuppressive activity (i.e., ability to inhibit [3H]thymidine incorporation into mitogen-stimulated lymphocytes) were localized in conceptus-conditioned culture medium: one corresponding to a high-molecular-weight glycoprotein (HMWG; Mr = 800-900 kDa), one corresponding to ovine trophoblast protein-1 (oTP-1), the antiluteolytic, interferon-like molecule of the sheep conceptus, and a third fraction containing a previously undescribed molecule with a molecular weight between 10 and 14 kDa. Both HMWG and oTP-1 inhibited lymphocyte proliferation in a dose-dependent manner. Delaying the addition of HMWG or oTP-1 until 24 h after the addition of phytohemagglutinin (PHA) reduced the degree of suppression relative to cultures where HMWG and oTP-1 were added at the time of PHA addition. Nonetheless, the molecules were still capable of inhibiting proliferation. Addition of human recombinant IL-2 (0-65 U/ml) to PHA-stimulated lymphocyte cultures could not reverse HMWG- or oTP-1-induced suppression of [3H]thymidine uptake by PHA-stimulated cells. Furthermore, treatment of lymphocytes with HMWG or oTP-1 suppressed IL-2-induced proliferation. Therefore, HMWG and oTP-1 affect both early and later events in the in vitro proliferative response of mitogen-stimulated lymphocytes, including responsiveness to IL-2.     

A macrophage-derived factor induced by alpha 1-acid glycoprotein that inhibits IL-1 comitogenic activity

After exposure to a concanavalin A (Con A)-unreactive variant of alpha 1-acid glycoprotein (AGP), macrophages released an inhibitor of interleukin-1 (IL-1) proliferative activity in the thymocyte comitogenic assay. This effect was observed with AGP concentrations above 100 micrograms/ml in the macrophage supernatant and would appear to be mediated by the macrophages, since native AGP had no activity on thymocyte proliferation. Preliminary physicochemical characterization showed that the factor was partially resistant to heating, undialyzable, and eluted with an apparent molecular mass of 50-100 kDa when subjected to Sephacryl S-200 chromatography. Murine IL-1 and human (h) recombinant (r) IL-1 were affected by this factor to the same extent. IL-1 and IL-2 co-induced thymocyte proliferation, which is mitogen-independent, was also inhibited, whereas hrIL-2 activity was not suppressed when assayed in thymocytes with PHA at a submitogenic concentration or in CTLL cells. The factor did not interfere with TNF alpha or hrIL-6 activity when tested against their specific cell line. These data indicate that the inhibitor may act specifically against IL-1 activity and further elucidate the possible role of AGP in the modulation of IL-1 activity via the secretion of an inhibitor.     

Human recombinant IL-1 receptor antagonist (IL-1Ra) inhibits leukotriene B4 generation from human monocyte suspensions stimulated by lipopolysaccharide (LPS).

The effect of human recombinant IL-1 receptor antagonist (hrIL-1Ra) on leukotriene B4 (LTB4) release was investigated in activated human monocyte cultures. To stimulate LTB4 generation, LPS was used as an agonist. Detection was performed with the highly sensitive radioimmunoassay method. The cells were treated with scalar concentrations using LPS at 1-1000 ng/ml for different periods of time. The greater LTB4 stimulation was found at LPS 100 ng/ml for 18 h incubation time. Preincubation of monocytes with cytochalasin B (CB) (5 micrograms/ml) for 15 min augmented the release of LTB4 when LPS was used. A dose-dependent inhibition was found when human monocytes were pretreated for 10 min with hrIL-1Ra at different concentrations (0.25-250 ng/ml) and then treated with LPS 100 ng/ml for 18 h. Maximum inhibition was observed at the highest concentration of hrIL-1Ra (250 ng/ml). Macrophages treated with a non-selective 5-lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), used at 10 microM, added 15 min before LPS 100 ng/ml, produce a dose-dependent inhibition of LTB4. Cells pretreated with arachidonic acid, at various concentrations (10(-9)-10(-5) M) for 10 min and then treated with LPS 100 ng/ml for 18 h, were also inhibited in a dose-dependent manner by hrIL-1Ra in their production of LTB4. The inhibition of LTB4 release by hrIL-1Ra, in LPS-stimulated human monocytes, may suggest an important modulatory role for this new cytokine (monokine) in inflammation and immunity and may hold future therapeutic implications for diseases involving LTB4 as a mediator.