Processing of interleukin-1 in cells of monocytic lineage is differentiation-dependent.

The interleukin-1 (IL-1) alpha and beta precursor proteins are processed and released from several cell types in the absence of a canonical signal peptide. To gain some insight into the mechanisms that allow the production of IL-1 alpha and beta, we have investigated by immunoprecipitation the synthesis, their release and processing in a promyeloblastic cell line of tumoral origin, U937, and in peripheral blood monocytes. We show that U937 monocytic cells, on induction with a tumor-promoting agent, synthesize and release into the culture medium proIL-1 beta but do not process it. Similarly, peripheral blood monocytes left in adherence for 24 h or longer, prior to addition of lipopolysaccharide, synthesize and release proIL-1 alpha and beta without detectable processing of either cytokine. Processing and release of IL-1 alpha and beta by peripheral blood monocytes can be observed when monocytes are left to adhere for periods less than 15 h before lipopolysaccharide addition. IL-1 alpha and beta show similar kinetics of release from the cells, suggesting the existence of a common mechanism regulating their secretion. Since peripheral blood monocytes left in adherence in the presence of lipopolysaccharide differentiate into macrophages, we conclude that release and processing of IL-1 can occur independently and that processing depends on the stage of differentiation of monocytes, i.e. only the monocytes at an early stage of differentiation produce 17-kDa IL-1 alpha and beta.     

Endotoxin-stimulated monocytes release multiple forms of IL-1 beta, including a proIL-1 beta form whose detection is affected by export

The processing and release of 31-kDa proIL-1 beta to the mature 17-kDa form of IL-1 beta are still poorly understood. To help elucidate the mechanisms involved in IL-1 beta processing and release, we measured IL-1 beta forms released from endotoxin-stimulated monocytes by immunoprecipitation of [35S]methionine-labeled protein, by Western blots, and by our recently developed ELISA specific for proIL-1 beta. Our studies demonstrate that in addition to the 17-kDa mature IL-1 beta, IL-1 beta is also released as 31-, 28-, and 3-kDa molecules. The 31-kDa-released form of proIL-1 beta represented 20-40% of the total released IL-1 beta, as measured by SDS-PAGE with densitometry. This released proIL-1 beta was susceptible to ICE processing; however, this proIL-1 beta was not detectable by either a mature or proIL-1 beta-specific ELISA, suggesting that release induces a conformational change. The ELISA inability to detect proIL-1 beta was not due to inadequate sensitivity or subsequent degradation in the ELISA. Furthermore, while immunoaffinity-purified cytosolic proIL-1 beta could complex the type II IL-1R, released proIL-1 beta did not. Finally, the absence of a band shift in nondenaturing gel electrophoresis excluded proIL-1 beta binding to another protein. These findings imply that IL-1 beta is exported from monocytes as 3-, 17-, 28-, and 31-kDa forms and that the released 31-kDa form differs from cytosolic proIL-1 beta.     

Virus infection activates IL-1 beta and IL-18 production in human macrophages by a caspase-1-dependent pathway.

Monocytes and macrophages play a significant role in host's defense system, since they produce a number of cytokines in response to microbial infections. We have studied IL-1 beta, IL-18, IFN-alpha/beta, and TNF-alpha gene expression and protein production in human primary monocytes and GM-CSF-differentiated macrophages during influenza A and Sendai virus infections. Virus-infected monocytes released only small amounts of IL-1 beta or IL-18 protein, whereas 7- and 14-day-old GM-CSF-differentiated macrophages readily produced these cytokines. Constitutive expression of proIL-18 was seen in monocytes and macrophages, and the expression of it was enhanced during monocyte/macrophage differentiation. Expression of IL-18 mRNA was clearly induced only by Sendai virus, whereas both influenza A and Sendai viruses induced IL-1 beta mRNA expression. Since caspase-1 is known to cleave proIL-1 beta and proIL-18 into their mature, active forms, we analyzed the effect of a specific caspase-1 inhibitor on virus-induced IL-1 beta and IL-18 production. The release of IL-1 beta and IL-18, but not that of IFN-alpha/beta or TNF-alpha, was clearly blocked by the inhibitor. Our results suggest that the cellular differentiation is a crucial factor that affects the capacity of monocytes/macrophages to produce IL-1 beta and IL-18 in response to virus infections. Furthermore, the virus-induced activation of caspase-1 is required for the efficient production of biologically active IL-1 beta and IL-18.     

Sera of lung cancer patients affect the release of Th1, Th2 and monocyte-derived cytokines, and the expression of IL-2Ralpha by normal, stimulated mononuclear cells

We have shown that the sera of lung cancer patients affect the response of ConA-stimulated normal peripheral blood mononuclear cells by decreasing the expression of IL-2Ralpha and inhibiting the release of IL-1beta and IL-2. A tendency to enhance the release of IL-6 was also observed. We conclude that an imbalance in the Th1/Th2 cytokine response, typical for cancer patients, may at least partly be related to soluble factors circulating in the patients' blood. We discuss a putative role of serum IL-10, IL-1ra, and soluble IL-2Ralpha in the effects observed.     

The inflammasome mediates UVB-induced activation and secretion of interleukin-1beta by keratinocytes

It has long been known that human keratinocytes are a potent source of the proinflammatory cytokines proIL-1alpha and -1beta[1], which are activated and released in response to UV irradiation [2]. However, the intracellular pathways, which regulate maturation and secretion of IL-1 in keratinocytes, are unknown. Here we show that the UVB-mediated enhancement of cytoplasmic Ca(2+) is required for activation of the IL-1beta-converting enzyme caspase-1 by the inflammasome, a multiprotein innate immune complex [3, 4]. Caspase-1 in turn activates proIL-1beta, and keratinocytes secrete the cytokine as well as inflammasome components. These results demonstrate the presence of a proIL-1beta-processing inflammasome in nonprofessional immune cells and the necessity of inflammasome components for the UVB-induced secretion of IL-1beta. This supports the concept that keratinocytes are important immuno-competent cells under physiological and pathological conditions [5].     

Priming of macrophages with lipopolysaccharide potentiates P2X7-mediated cell death via a caspase-1-dependent mechanism, independently of cytokine production.

ATP stimulation of cell surface P2X7 receptors results in cytolysis and cell death of macrophages. Activation of this receptor in bacterial lipopolysaccharide (LPS)-activated macrophages or monocytes also stimulates processing and release of the cytokine interleukin-1beta(IL-1beta) through activation of caspase-1. The cytokine interleukin 18 (IL-18) is also cleaved by caspase-1 and shares pro-inflammatory characteristics with IL-1beta. The objective of the present study was to test the hypothesis that IL-1beta, IL-18, and/or caspase-1 activation contribute directly to macrophage cell death induced by LPS and ATP. Macrophages were cultured from normal mice or those in which genes for the P2X7 receptor, IL-1beta, IL-1alpha, IL-18, or caspase-1 had been deleted. Our data confirm the importance of the P2X7 receptor in ATP-stimulated cell death and IL-1beta release from LPS-primed macrophages. We demonstrate that prolonged stimulation with ATP leads to cell death, which is partly dependent on LPS priming and caspase-1, but independent of cytokine processing and release. We also provide evidence that LPS priming of macrophages makes them highly susceptible to the toxic effects of brief exposure to ATP, which leads to rapid cell death by a mechanism that is dependent on caspase-1 but, again, independent of cytokine processing and release.     

ATP acts as an agonist to promote stimulus-induced secretion of IL-1 beta and IL-18 in human blood

Cultured monocytes and macrophages stimulated with LPS produce large quantities of proIL-1beta, but release little mature cytokine to the medium. The efficiency at which the procytokine is converted to its active 17-kDa species and released extracellularly is enhanced by treating cytokine-producing cells with a secretion stimulus such as ATP or nigericin. To determine whether this need for a secretion stimulus extends to blood, individual donors were bled twice daily for 4 consecutive days, and the collected blood samples were subjected to a two-step IL-1 production assay. LPS-activated blood samples generated cell-free IL-1beta, but levels of the extracellular cytokine were greatly increased by subsequent treatment with ATP or nigericin. Specificity and concentration requirements of the nucleotide triphosphate effect suggests a P2X(7) receptor involvement. Quantities of IL-1beta generated by an individual donor's blood in response to the LPS-only and LPS/ATP stimuli were relatively consistent over the 4-day period. Between donors, consistent differences in cytokine production capacity were observed. Blood samples treated with ATP also demonstrated enhanced IL-18 production, but TNF-alpha levels decreased. Among leukocytes, monocytes appeared to be the most affected cellular targets of the ATP stimulus. These studies indicate that an exogenous stimulus is required by blood for the efficient production of IL-1beta and IL-18, and suggest that circulating blood monocytes constitutively express a P2X(7)-like receptor.     

Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1

Caspases are intracellular proteases that cleave substrates involved in apoptosis or inflammation. In C. elegans, a paradigm for caspase regulation exists in which caspase CED-3 is activated by nucleotide-binding protein CED-4, which is suppressed by Bcl-2-family protein CED-9. We have identified a mammalian analog of this caspase-regulatory system in the NLR-family protein NALP1, a nucleotide-dependent activator of cytokine-processing protease caspase-1, which responds to bacterial ligand muramyl-dipeptide (MDP). Antiapoptotic proteins Bcl-2 and Bcl-X(L) bind and suppress NALP1, reducing caspase-1 activation and interleukin-1beta (IL-1beta) production. When exposed to MDP, Bcl-2-deficient macrophages exhibit more caspase-1 processing and IL-1beta production, whereas Bcl-2-overexpressing macrophages demonstrate less caspase-1 processing and IL-1beta production. The findings reveal an interaction of host defense and apoptosis machinery.     

Pannexin-1 mediates large pore formation and interleukin-1beta release by the ATP-gated P2X7 receptor

P2X(7) receptors are ATP-gated cation channels; their activation in macrophage also leads to rapid opening of a membrane pore permeable to dyes such as ethidium, and to release of the pro-inflammatory cytokine, interleukin-1beta (IL-1beta). It has not been known what this dye-uptake path is, or whether it is involved in downstream signalling to IL-1beta release. Here, we identify pannexin-1, a recently described mammalian protein that functions as a hemichannel when ectopically expressed, as this dye-uptake pathway and show that signalling through pannexin-1 is required for processing of caspase-1 and release of mature IL-1beta induced by P2X(7) receptor activation.     

Essential role for Ca2+ in regulation of IL-1beta secretion by P2X7 nucleotide receptor in monocytes,macrophages, and HEK-293 cells

Interleukin (IL)-1beta is a proinflammatory cytokine that elicits the majority of its biological activity extracellularly, but the lack of a secretory signal sequence prevents its export via classic secretory pathways. Efficient externalization of IL-1beta in macrophages and monocytes can occur via stimulation of P2X7 nucleotide receptors with extracellular ATP. However, the exact mechanisms by which the activation of these nonselective cation channels facilitates secretion of IL-1beta remain unclear. Here we demonstrate a pivotal role for a sustained increase in cytosolic Ca2+ to potentiate secretion of IL-1beta via the P2X7 receptors. Using HEK-293 cells engineered to coexpress P2X7 receptors with mature IL-1beta (mIL-1beta), we show that activation of P2X7 receptors results in a rapid secretion of mIL-1beta by a process(es) that is dependent on influx of extracellular Ca2+ and a sustained rise in cytosolic Ca2+. Moreover, reduction in extracellular Ca2+ attenuates approximately 90% of P2X7 receptor-mediated IL-1beta secretion but has no effect on enzymatic processing of precursor IL-1beta (proIL-1beta) to mIL-1beta by caspase-1. Similar experiments with THP-1 human monocytes and Bac1.2F5 murine macrophages confirm the unique role of Ca2+ in P2X7 receptor-mediated secretion of IL-1beta. In addition, we report that cell surface expression of P2X7 receptors in the absence of external stimulation also results in enhanced release of IL-1beta and that this can be repressed by inhibitors of P2X7 receptors. We clarify an essential role for Ca2+ in ATP-induced IL-1beta secretion and indicate an additional role of P2X7 receptors as enhancers of the secretory apparatus by which IL-1beta is released.     

Haptoglobin and the Th1/Th2 balance: hints from in vitro and in vivo studies

A variety of immunomodulatory effects have previously been attributed to haptoglobin (Hp). These are supposed to be partly mediated through binding of Hp to CD11b. In the present study, we assessed its effects on T-helper (Th) cytokine production following both in vitro and in vivo stimulation of T-cells. Hp exhibits a dose-dependent inhibitory effect on human T lymphocyte release of the Th2 cytokines (IL-4, IL-5, IL-10 and IL-13) in vitro, whereas it has no clear effect on Th1 cytokine (IL-2 and IFN-gamma) release. When administered an anti-CD3 monoclonal antibody, Hp knockout mice produced more IL-4 and less IFN-gamma than did their wild-type litter-mates. Our findings imply that Hp may be regarded as a regulator of the Th1/Th2 balance in both human and murine immune systems.     

Prointerleukin-18 is activated by meprin beta in vitro and in vivo in intestinal inflammation

Interleukin-18 (IL-18), a pro-inflammatory cytokine, is a key factor in inflammatory bowel disease (IBD). Caspase-1 activates this cytokine, but other proteases are likely involved in maturation. Because meprin metalloproteinases have been implicated in IBD, the interaction of these proteases with proIL-18 was studied. The results demonstrate that the meprin beta subunit of meprins A and B cleaves proIL-18 into a smaller 17-kDa product. The cleavage is at the Asn51-Asp52 bond, a site C-terminal to caspase-1 cleavage. The cleavage occurred in vitro with a Km of 1.3 microm and in Madin-Darby canine kidney cells transfected with meprin beta when proIL-18 was added to the culture medium. The product of meprin B cleavage of proIL-18 activated NF-kappaB in EL-4 cells, indicating that it was biologically active. To determine the physiological significance of the interactions of meprins with proIL-18, an experimental model of IBD was produced by administering dextran sulfate sodium (DSS) to wild-type and meprin beta knock-out (betaKO) mice, and the serum levels of active IL-18 were determined. DSS-treated meprin betaKO mice had lower levels of the active cytokine in the serum compared with wild-type mice. Furthermore, in meprin alphaKO mice, which express meprin beta but not alpha, active IL-18 was elevated in the serum of DSS-treated mice compared with wild-type mice, indicating that the meprin isoforms have opposing effects on the IL-18 levels in vivo. This study identifies proIL-18 as a biologically important substrate for meprin beta and implicates meprins in the modulation of inflammation.     

Testosterone and estrogen differently effect Th1 and Th2 cytokine release following trauma-haemorrhage.

The object of the study was to determine whether male and female sex steroids produce divergent effects on Th1 and Th2 cytokine release following trauma-haemorrhage. Recent studies indicate that androgens are responsible for the depressed splenocyte Th1 cytokine release in males following trauma-haemorrhage. In contrast, female mice maintain their Th1 cytokine release capacity following trauma-haemorrhage. Nonetheless, the effect of male and female sex steroids on Th1 and Th2 cytokine release following trauma-haemorrhage remains unknown. Male C3H/HeN mice were castrated and treated with pellets containing either vehicle, 5alpha-dihydrotestosterone (DHT), 17beta-estradiol (estradiol), or a combination of both steroid hormones, for 14 days prior to soft-tissue trauma (i.e. laparotomy) and haemorrhagic shock (35+/-5 mmHg for 90 min followed by adequate fluid resuscitation) or sham operation. Untreated male and female mice, as well as DHT treated female mice, served as control groups. Twenty-four hours later the animals were sacrificed, plasma obtained and splenocytes harvested. Plasma DHT and estradiol levels in treated animals were comparable with intact male and female mice, respectively. A significant depression of splenocyte Th1 cytokines, i.e. IL-2, IFN-gamma, was observed in DHT treated castrated animals, DHT treated females, and untreated males following trauma-haemorrhage, as opposed to maintained Th1 cytokine release in estradiol treated and estradiol/DHT treated castrated animals and females. The release of the anti-inflammatory cytokine IL-10 was markedly increased in DHT treated mice and males subjected to trauma-haemorrhage compared to shams, but decreased in estrogen treated mice and females under such conditions. These results suggest that male and female sex steroids differentially affect the release of Th1 and Th2 cytokines following trauma-haemorrhage and should be further studied for their potential to modulate splenocyte function in trauma victims.     

Osteopontin-dependent regulation of Th1 and Th17 cytokine responses in Trypanosoma cruzi-infected C57BL/6 mice

Osteopontin (OPN) is a multifunctional protein participating in the regulation of different Th cell lineages and critically involved in the initiation of immune responses to diverse pathogens. Our study goal was to verify whether OPN helps modulate the protective Th1 and Th17 cytokine responses in C57BL/6 mice infected with Trypanosoma cruzi, the etiological agent of Chagas disease. Parasite infection induced OPN release from murine macrophages in vitro and acute Chagas mice displayed enhanced serum levels of this cytokine at the peak of parasitemia. Upon administration of a neutralizing anti-OPN antibody, recently infected mice presented lower Th1 and Th17 responses, increased parasitemia and succumbed earlier and at higher rates to infection than non-immune IgG-receiving controls. The anti-OPN therapy also resulted in reduced circulating levels of IL-12 p70, IFN-γ, IL-17A and specific IgG_2a antibodies. Furthermore, antibody-mediated blockade of OPN activity abrogated the ex vivo production of IL-12 p70, IFN-γ and IL-17A, while promoting IL-10 secretion, by spleen macrophages and CD4⁺ T cells from T. cruzi-infected mice. Th1 and Th17 cytokine release induced by OPN preferentially involved the α_vβ₃ integrin OPN receptor, whereas concomitant down-modulation of IL-10 production would mostly depend on OPN interaction with CD44. Our findings suggest that, in resistant C57BL/6 mice, elicitation of protective Th1 and Th17 cytokine responses to T. cruzi infection is likely to be regulated by endogenous OPN.     

IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells

Murine IL-10 (cytokine synthesis inhibitory factor) inhibits cytokine production by Th1 cell clones when they are activated under conditions requiring the presence of APC. By preincubating APC with IL-10, we demonstrate that IL-10 acts principally on APC to inhibit IFN-gamma production by Th1 clones. Moreover, IL-10 is not active when Th1 cells are stimulated with glutaraldehyde-fixed APC, which also indicates that its action involves regulation of APC function. Furthermore, IL-10 inhibits cytokine synthesis by Th1 cells stimulated with the super-antigen Staphylococcus enterotoxin B, which does not appear to require processing. Flow microfluorimetry purified splenic or peritoneal B cells and macrophages, and B cell and macrophage cell lines can present Ag to Th1 clones. However, IL-10 acts only on sorted macrophages and the macrophage cell line to suppress IFN-gamma production by Th1 clones. IL-10 does not show this effect when B cells are used as APC. In contrast, IL-10 does not impair the ability of APC to stimulate cytokine production by Th2 cells. IL-10 does not decrease IFN-gamma-induced I-Ad levels on a macrophage cell line. Inasmuch as IL-10 also inhibits IL-2-induced IFN-gamma production by Th1 cells in an Ag-free system requiring only the presence of accessory cells, these data suggest that IL-10 may inhibit macrophage accessory cell function which is independent of TCR-class II MHC interactions.     

Haptoglobin and the Th1/Th2 balance: hints from in vitro and in vivo studies

Abstract

A variety of immunomodulatory effects have previously been attributed to haptoglobin (Hp). These are supposed to be partly mediated through binding of Hp to CD11b. In the present study, we assessed its effects on T-helper (Th) cytokine production following both in vitro and in vivo stimulation of T-cells. Hp exhibits a dose-dependent inhibitory effect on human T lymphocyte release of the Th2 cytokines (IL-4, IL-5, IL-10 and IL-13) in vitro, whereas it has no clear effect on Th1 cytokine (IL-2 and IFN-γ) release. When administered an anti-CD3 monoclonal antibody, Hp knockout mice produced more IL-4 and less IFN-γ than did their wild-type litter-mates. Our findings imply that Hp may be regarded as a regulator of the Th1/Th2 balance in both human and murine immune systems.     

Th1 and Th2 cytokine immunomodulation by gangliosides in experimental autoimmune encephalomyelitis

In experimental autoimmune encephalomyelitis, a classical model for multiple sclerosis, the cytokines provide the necessary signals to activate specific T cells for self-antigens. Gangliosides have multiple immunomodulatory activities, decreasing the lymphoproliferative responses and modulating cytokine production. Here, we tested the effects of gangliosides on the switching of Th1 to Th2 cytokine expression, in spleen cells obtained from Lewis rats during the acute phase of EAE, and after recovery from the disease. For this purpose, total RNA from spleen cells was isolated and submitted to RT-PCR to investigate Th1 (IL-2, TNF-alpha, and IFN-gamma) and Th2/Th3 (IL-10 and TGF-beta) cytokine gene expression. Results demonstrate that the group treated with gangliosides displays mild disease, with low expression of IFN-gamma mRNA and high TGF-beta mRNA expression. We conclude that the gangliosides may modulate Th1 cells by the synthesis of cytokines shifting the profile to the Th2/Th3 phenotype.