Interleukin-16 stimulates the expression and production of pro-inflammatory cytokines by human monocytes

Interleukin-16 (IL-16) acts as a chemoattractant for CD4+ cells, as a modulator of T-cell activation, and plays a key role in asthma. This report describes the cytokine-inducing effects of IL-16 on total peripheral blood mononuclear cells (PBMC) and PBMC subpopulations. While CD4+ T lymphocytes did not secrete cytokines in response to rhIL-16, CD14+ CD4+ monocytes and maturing macrophages secrete IL-1beta, IL-6, IL-15 and tumour necrosis factor-alpha (TNF-alpha) upon rhIL-16 stimulation. The mRNA species for these four cytokines were detected as early as 4 hr post-stimulation, with protein being secreted by 24 hr. Secretion of IL-1beta and IL-6 by total PBMC was dose dependent, with maximal secretion being observed using 50 ng/ml rhIL-16. However, for IL-15 or TNF-alpha maximal secretion by total PBMC occurred with all concentrations between 5 ng/ml to 500 ng/ml rhIL-16. Purified monocytes/macrophages secreted maximal concentrations of all four cytokines in the presence of 500 ng/ml rhIL-16, except for monocytes where maximal secretion of IL-15 was, interestingly, observed with only 50 ng/ml rhIL-16. The use of higher concentrations of rhIL-16 (1000 ng/ml) inhibited secretion of all four cytokines. While these IL-16-induced cytokines are likely to be involved in the immune system's response to antigen, the data suggest that IL-16 may play a key role in initiating and/or sustaining an inflammatory response.     

Recombinant human interleukin-1 beta primes human polymorphonuclear leukocytes for stimulus-induced myeloperoxidase release

Recombinant human Interleukin-1 (rhIL-1) beta was found to enhance stimulus-induced granule exocytosis from human polymorphonuclear leukocytes (PMNs). PMNs were incubated with rhIL-1 beta and then stimulated with either heat-aggregated IgG (Hagg) or N-formyl-methionyl leucylphenylalanine (FMLP). The release of the azurophil enzyme myeloperoxidase (MPO) was measured. Low concentrations of stimuli (10 micrograms/ml Hagg, 2.5 X 10(-9) M FMLP) did not stimulate degranulation in the absence of rhIL-1 beta. However, such concentrations elicited marked degranulation from PMNs preincubated with rhIL-1 beta (0.2-100 ng/ml). The enhancement of degranulation was dependent on the concentration of rhIL-1 beta employed and on the period of incubation. In other experiments, the effect of rhIL-1 beta on the PMN oxidative response was determined. rhIL-1 beta did not directly stimulate the production of superoxide anions or enhance the oxidative response to Hagg or FMLP. It is suggested that in rheumatoid joints, IL-1 beta may potentiate PMN degranulation, but not their oxidative response.     

Immunoglobulin D enhances the release of tumor necrosis factor-alpha, and interleukin-1 beta as well as interleukin-1 receptor antagonist from human mononuclear cells.

Immunoglobulin D (IgD) is normally present in only low concentrations in serum. In the hyper-IgD and periodic fever syndrome (HIDS), however, serum levels exceed 140 mg/l. This syndrome is further characterized by recurrent inflammatory febrile attacks together with an acute phase response and appearance of cytokines in the circulation. The role of IgD in the pathogenesis of HIDS and its relation to the increased cytokine concentrations is unclear. Therefore, we tested whether IgD, IgG and alpha 1-acid glycoprotein (AGP) isolated from human serum influence the synthesis of interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), and IL-1ra, as measured by specific radioimmunoassays, in human peripheral blood mononuclear cells (PBMC). Incubation of PBMC with IgD and AGP for 24 hr led to increased release of IL-1 beta, TNF-alpha, and IL-lra. The magnitude of stimulation of IgD exceeded that of AGP; the effect by IgD was dose-dependent and showed a 30-fold (TNF-alpha) to almost 150-fold (IL-1 beta) increase at the highest concentration (50 mg/l), while AGP (750 micrograms/ml) only increased the cytokine secretion fourfold (TNF-alpha) to almost 30-fold (IL-1 beta). The effect of IgD on IL-1ra was less dramatic but a fivefold increase was observed at 50 mg/l compared with a 2.5-fold increase with AGP. IgD potentiated the effect of lipopolysaccharide (LPS) on secretion of both IL-1 beta and TNF-alpha, although the effect was most apparent for TNF-alpha. Apart from inducing IL-1ra synthesis, IgG did not influence cytokine release in human PBMC. These data indicate that IgD is a potent inducer of TNF-alpha, IL-1 beta and IL-1ra and thus may contribute to the pathogenesis of HIDS.     

Intracerebroventricular administration of a specific IL-1 receptor antagonist blocks food and water intake suppression induced by interleukin-1 beta.

Intracerebroventricular (ICV) microinfusion of recombinant human interleukin-1 beta (rhIL-1 beta, 1.0 ng/rat) suppressed the short-term (2 h) food and water intakes in rats. ICV infusion of recombinant human IL-1 receptor antagonist (rhIL-1ra) had no effect on food or water intake. Concomitant ICV infusion of rhIL-1 beta (1.0 ng/rat) and increasing concentrations of rhIL-1ra (25 to 1000 ng/rat) resulted in a dose-dependent inhibition of the short-term food and water intake suppression induced by rhIL-1 beta. The highest doses of rhIL-1ra (500 and 1000 ng/rat) completely blocked the food and water intake suppression induced by rhIL-1 beta. This suggests specificity of IL-1 beta suppression of short-term feeding and drinking by a direct action in the central nervous system (CNS). This ability of rhIL-1ra may have potential therapeutic implications in acute and chronic pathological processes associated with increased levels of IL-1 and appetite suppression.     

Alpha 1-acid glycoprotein potentiates lipopolysaccharide-induced secretion of interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha by human monocytes and alveolar and peritoneal macrophages.

Although the physiological role of alpha 1-acid glycoprotein (AGP), an acute-phase protein, is poorly understood, several lines of evidence support a modulatory action on the immune response. In this study, we investigated the effect of AGP on the production of interleukin (IL)-1 beta, IL-6 and tumor necrosis factor (TNF)-alpha by human monocytes, macrophages and the monocytic THP-1 cell line. AGP significantly enhanced (2- to 7-fold) the production of these cytokines in monocytes induced by suboptimal concentrations of lipopolysaccharide [E. coli lipopolysaccharide (LPS): 100 ng/ml] in serum-free conditions, whereas it had little or no effect in the absence of LPS. The potentiating effect of AGP was inhibited by specific antibodies. It was concentration dependent and the greatest enhancement was observed with 250-500 micrograms/ml. Moreover, AGP only potentiated the effect of suboptimal concentrations of LPS. AGP did not alter the time course of LPS-induced IL-1 beta, IL-6 or TNF-alpha secretion. AGP acts as a co-inducer and could also potentiate cytokine secretion triggered by Neisseria meningitidis LPS and muramyl dipeptide. The glycan moiety of AGP did not seem to be involved in its potentiating effect, since both its major glycoforms and asialo-AGP potentiated the effect of LPS to the same extent as native AGP. Possible differences in the effect of AGP according to cell maturation were investigated using isolated human macrophages: AGP potentiated LPS-induced cytokine production by both peritoneal and alveolar macrophages. These data suggest that AGP can modulate monocyte/macrophage functions, thereby contributing to the amplification and regulation of immune and inflammatory responses.     

Modulation of IL-1 alpha, IL-1 beta, and 25K Mr non-IL-1 activity released by human mononuclear cells

To determine if the release of IL-1 alpha and IL-1 beta by cultured PBMC could be independently modulated by different exogenous stimuli, we examined the effect of LPS, IFN gamma, latex beads, and indomethacin on the release of IL-1 alpha and IL-1 beta. PBMC culture supernatants were fractionated by Sephacryl-S-200 column chromatography or HPLC (TSK G3000SW), and each fraction was tested for thymocyte mitogenic activity in the presence or absence of preincubation with anti-IL-1 alpha or anti IL-1 beta monoclonal antibody (mAb) and for the presence of IL-1 alpha or IL-1 beta protein by ELISA. In all experiments, thymocyte mitogenic activity not neutralizable by anti-IL-1 alpha or anti-IL-1 beta mAb was detected in the 25K Mr range, which ranged from 12 to 50% of the total thymocyte mitogenic activity released, depending on the stimuli. Cultured PBMC from 95% of individuals release thymocyte mitogenic activity in the absence of exogenous stimuli, which was increased 1.3-to 7-fold by lopopolysaccharide (LPS) (25-50 micrograms/ml). All of this increased activity was due to increased release of IL-1 beta and non-IL-1 thymocyte mitogenic activity, with no change in the total amount of IL-1 alpha released. Indomethacin (0.1 microgram/ml) induced release of increased thymocyte mitogenic activity of 1.3- to 1.4-fold over unstimulated cultures. All of this increased activity was due to increased release of IL-1 alpha and non-IL-1 activity with a concomitant decrease in IL-1 beta release. Interferon gamma (40-100 U/ml) increased the amount of IL-1 alpha and decreased IL-1 beta and non-IL-1 activity released, resulting in no overall change in the total amount of thymocyte mitogenic activity. Molecular weight fractionation of the PBMC culture supernatants revealed that thymocyte mitogenic activity eluting in the 25K Mr range was not due to IL-1 alpha or IL-1 beta. With certain culture conditions, thymocyte mitogenic activity was detected in the 30-40K Mr range. PBMC cultured with LPS and latex beads in the absence of serum released 30-40K Mr IL-1 alpha, as well as 17K Mr IL-1 alpha and 17K Mr IL-1 beta. PBMC cultured in 2% fetal calf serum (FCS) alone from some donors released only 30-40K Mr thymocyte mitogenic activity. Both IL-1 alpha and IL-1 beta protein was detected by ELISA in this Mr range but only the IL-1 alpha was bioactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of fibronectin synthesis by interleukin-1 and interleukin-6 in rat hepatocytes.

The authors have observed previously that recombinant human interleukin-1 (rhIL-1) administered into rats increased plasma fibronectin (Fn) level concomitant with the increase of Fn in the liver. Because IL-1 induces interleukin-6 (IL-6) in certain cell types, the IL-1 effect might be mediated by IL-6. To evaluate this possibility, the effect of recombinant human interleukin-6 (rhIL-6), rhIL-1 alpha, and rhIL-1 beta on Fn synthesis in cultured rat hepatocytes was studied. It was shown that rhIL-6 increased Fn synthesis in hepatocytes, in contrast, rhIL-1 alpha, rhIL-I beta and TNF did not have any effect on Fn synthesis. When we studied the interaction of IL-1 and IL-6, IL-1 did not exhibit any synergistic effect with IL-6. Conditioned medium (CM) from rhIL-1 stimulated peripheral blood monocytes (PBM) increased the Fn synthesis, and its activity was neutralized significantly by anti-rhIL-6 antibodies. The CM from rhIL-1-stimulated PBM was analyzed by enzyme linked immunosorbent assay and revealed the increase of IL-6. Furthermore, it was found that intraperitoneal administration of rhIL-1 induced IL-6 into blood. The administration of rhIL-6 into rats increased circulating Fn levels. These results strongly suggest that the in vivo effect of IL-1 on Fn synthesis is mediated by IL-6.     

Marijuana components stimulate human peripheral blood mononuclear cell secretion of interferon-gamma and suppress interleukin-1 alpha in vitro.

We investigated the in vitro effects of both psychoactive and nonpsychoactive marijuana components on leukocyte secretion of the immunoregulatory cytokines interleukin-1 alpha (IL-1), tumor necrosis factor alpha (TNF), interferon-gamma (IFN) and interleukin-2 (IL-2). Psychoactive delta-9-tetrahydrocannabinol (THC) and nonpsychoactive cannabidiol (CBD) were added to cultures of mitogen-activated human peripheral blood mononuclear cells (PBMC) and the concentrations of IL-1, TNF, IFN and IL-2 in culture supernatants were measured by ELISA systems. Concentrations of THC and CBD, comparable to plasma levels found after smoking marijuana (10-100 ng/ml), increased the concentration of measurable IFN (139 and 68%), while high concentrations of both cannabinoids (5-20 micrograms/ml) completely blocked synthesis and/or release of this cytokine. CBD was also shown to decrease the measurable quantity of both IL-1 and TNF. In contrast to the effects on IFN, IL-1 and TNF, both cannabinoids, had no effect on IL-2 secretion. This report suggests that both psychoactive and nonpsychoactive components of marijuana are immunomodulating and can potentially alter cytokine secretion of human PBMC.     

Interleukin-1 alpha and interleukin-1 beta in preterm and term human parturition.

Interleukin-1 (IL-1) has been implicated in the mechanism of human parturition in the setting of infection. The purpose of this study was to determine the effect of labor (term and preterm) and microbial invasion of the amniotic cavity on amniotic fluid (AF) concentrations IL-1 alpha and IL-1 beta. AF was retrieved by transabdominal amniocentesis from the following groups of women: midtrimester genetic amniocentesis (16 to 18 wk) (N = 15), preterm labor with intact membranes (21 to 36 wk) with or without infection (N = 72), preterm premature rupture of membranes (PROM) (N = 88), and term not in labor or in active labor with or without infection (N = 58). AF was cultured for aerobic and anaerobic bacteria as well as Mycoplasmas. IL-1 was measured with a commercially available immunoassay validated for AF (sensitivity: IL-1 alpha, 157 pg/ml; IL-1 beta, 50 pg/ml). All women at midtrimester had undetectable AF IL-1 alpha and IL-1 beta. Among women in preterm labor with positive AF cultures, IL-1 alpha and IL-1 beta were detectable in the AF in 86.6% (13/15) and 100% (15/15), respectively. In contrast, all women with negative AF cultures without labor (N = 36) had undetectable AF IL-1 alpha concentrations and 52.7% (19/36) had undetectable AF IL-1 beta concentrations. Histopathological chorioamnionitis was present in 92.8% (13/14) of patients who had positive AF cultures and detectable IL-1 in the AF. IL-1 was significantly higher in patients with preterm PROM, labor, and positive AF cultures than in the other subgroups of patients with preterm PROM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neutral endopeptidase (EC 3.4.24.11) does not hydrolyze recombinant human interleukin-1 beta.

We have previously shown that neutral endopeptidase (NEP; EC 3.4.24.11) regulates neuropeptide-induced responses. Recently, Pierart et al. reported that NEP degraded purified interleukin-1 (IL-1) using thymocyte proliferation assay. Since IL-1 is an important cytokine in the immune response and inflammation, we have assessed whether NEP hydrolyzes recombinant human IL-1 beta using three assay systems (bioassay, immunoassay, and HPLC analysis). NEP on the NALM-6 cells (both intact cells and the solubilized plasma membrane fraction) efficiently hydrolyzed Met5-enkephalin and substance P. However, NEP did not significantly decrease the amount of rhIL-1 beta assessed by the growth inhibitory activity of a human melanoma, by the immunoassay, or by the direct analysis on HPLC. Therefore, we conclude that NEP does not significantly hydrolyze rhIL-1 beta. Our results suggest that, in contrast to the regulatory role of NEP in neuropeptide-induced responses, NEP is not a regulatory enzyme for IL-1-induced responses.     

Interleukin-4 inhibits secretion of interleukin-1beta in the response of human cells to mycobacterial heat shock proteins.

Cellular activation induced by Mycobacterium bovis bacillus Calmette-Guérin (BCG) and heat shock proteins (HSP) leads to the production of proinflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-6. In this study, we found that IL-4 significantly suppressed IL-1beta secretion induced by BCG and the 70- and 65-kDa HSP. When exogenous recombinant human IL-4 was added to human mononuclear cells, a dose- and time-related inhibition of the 70-kDa HSP- and BCG-induced IL-1beta secretion was observed. IL-1beta secretion was maximally inhibited at 24 h of culture, and this inhibitory effect was sustained at a later time point of culture (120 h). In addition, IL-2, another T-cell-derived cytokine acting on monocytes, had no effect on IL-1beta secretion induced by either BCG or the 70-kDa HSP, indicating that in these experiments not all cytokines could immunoregulate IL-1beta secretion. This inhibitory effect was not due to a cytotoxic effect of IL-4, since the viabilities of human mononuclear cells were comparable in the presence and absence of IL-4. IL-4 was also able to inhibit the secretion of IL-1beta by mycobacterium-stimulated cells from three rheumatoid arthritis patients. This inhibitory effect of IL-4 was reversed with a blocking anti-IL-4 antibody. Finally, IL-4 inhibited IL-6 secretion by mycobacterium-activated human cells. These results suggest that IL-4 may be important in the regulation of the immune response to mycobacterial antigens.     

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.     

Effect of human interleukin-2 from different sources on lymphocyte and airway beta-adrenoceptor function

The effect of recombinant human interleukin-2 (rh IL-2, Genzyme, yeast derived) on the beta-adrenoceptor function of human peripheral blood mononuclear cells (PBMC), guinea pig splenic lymphocytes and isolated guinea pig tracheal spirals was investigated. Rh IL-2 (Genzyme, yeast derived) induces a dose dependent inhibition of the isoprenaline-stimulated cAMP production in PBMC and splenic lymphocytes after a two hour preincubation period. The inhibition is significant at 0.01 U/ml IL-2 and reaches a maximum at 1 U/ml amounting 81 +/- 8% and 76 +/- 6% for human PBMC and guinea pig splenic lymphocytes respectively. The sensitivity of isolated guinea pig tracheal spirals to isoprenaline is also significantly decreased after a two hour preincubation period with 1 U/ml rh IL-2 (Genzyme, yeast derived). In contrast, rh IL-2 (Cetus, bacteria derived) does not affect the beta-adrenoceptor function of human PBMC, guinea pig splenic lymphocytes and isolated tracheal spirals, after a two-hour preincubation period. Furthermore, human cell-line derived IL-2 (Jurkat, Genzyme) also does not influence human PBMC beta-adrenoceptor function. It can therefore be concluded that IL-2 does not affect lymphocyte and airway beta-adrenoceptor function after a two hour preincubation period. The inhibition of beta-adrenoceptor function by yeast derived rh IL-2 (Genzyme) is therefore probably not related to IL-2.     

Comparative responses of human and rabbit interleukin-1 in vivo: effect of a recombinant interleukin-1 receptor antagonist.

The ability of recombinant human and rabbit interleukin-1 alpha (IL-1 alpha) in inducing inflammatory responses in rabbit skin were compared. Intradermal (i.d.) injections of recombinant human IL-1 alpha and recombinant rabbit IL-1 alpha induced intense accumulation of 111In-labelled neutrophils which was dependent on the dose of the cytokines administered. Both forms of IL-1 alpha induced very small levels of plasma protein leakage. Co-injection of the cytokines with the mRNA synthesis inhibitor actinomycin D (Act D) attenuated the number of neutrophils accumulating in response to both human and rabbit forms of IL-1 alpha. Local injection of a recombinant human IL-1 receptor antagonist (IL-1Ra) caused a dose-dependent inhibition of local inflammatory responses initiated by human and rabbit IL-1 alpha s well as rabbit IL-1 beta indicating the species cross-reactivity of the antagonist. IL-1Ra was selective for IL-1 in rabbit skin, as responses induced by C5ades Arg and formyl-methionyl-leucyl-phenylalanine (FMLP) were not inhibited. IL-1Ra significantly inhibited the IL-1-induced neutrophil accumulation only when co-injected with the cytokine. The local administration of the antagonist 30 min after rabbit IL-1 alpha failed to inhibit the inflammatory response. These results suggest that the in vivo events leading to the accumulation of neutrophils in response to IL-1 alpha are rapidly initiated.     

Human recombinant interleukin-1 receptor antagonist (hrIL-1ra) enhances the stimulatory effect of interleukin-2 on natural killer cell activity against MOLT-4 target cells.

Interleukin-1 (IL-1) is considered an enhancer of host defence against malignancies. Patients with different diseases, including cancer patients with large tumour burdens, have demonstrated a reduced production of IL-1 from circulating leukocytes, in vitro. There are many naturally occurring substances which inhibit IL-1 activity aspecifically. Recently an interleukin-1 receptor antagonist (IL-1ra) has been discovered, which is secreted by human macrophages and is structurally similar to IL-1 beta (26% homology). The pretreatment of human peripheral blood mononuclear cells (PBMC) with hrIL-1ra (0.25-250 ng/ml) inhibits IL-1 alpha or IL-1 beta and enhances, in a dose-dependent manner, the stimulatory effect of IL-2 on their natural killer (NK) activity against a lymphoid cell line MOLT-4. The enhancing effect of IL-1ra on IL-2 activity was similar to that provoked by IL-1 beta. However, when IL-1ra was used alone without IL-2, no stimulatory effect was found compared with the control. In our data we show that a member of the IL-1 family, IL-1ra, has a significant effect on IL-2-stimulated NK activity against the MOLT-4 cell line. These studies provide new evidence of the biological potential of IL-1ra since this new protein enhances IL-2 activity on NK cells.