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Naïve CD8 + T lymphocytes exposed to certain inflammatory cytokines undergo proliferation and display increased sensitivity to antigens. Such 'cytokine priming' can promote the activation of potentially autoreactive and antitumor CD8 + T cells by weak tissue antigens and tumor antigens. To elucidate the molecular mechanisms of cytokine priming, naïve PMEL-1 TCR transgenic CD8 + T lymphocytes were stimulated with IL-15 and IL-21, and chromatin accessibility was assessed using the assay for transposase-accessible chromatin (ATAC) sequencing. PMEL-1 cells stimulated by the cognate antigenic peptide mgp100 25-33 served as controls. Cytokine-primed cells showed a limited number of opening and closing chromatin accessibility peaks compared to antigen-stimulated cells. However, the ATACseq peaks in cytokine-primed cells substantially overlapped with those of antigen-stimulated cells and mapped to several genes implicated in T cell signaling, activation, effector differentiation, negative regulation and exhaustion. Nonetheless, the expression of most of these genes was remarkably different between cytokine-primed and antigen-stimulated cells. In addition, cytokine priming impacted the expression of several genes following antigen stimulation in a synergistic or antagonistic manner. Our findings indicate that chromatin accessibility changes in cytokine-primed naïve CD8 + T cells not only underlie their increased antigen responsiveness but may also enhance their functional fitness by reducing exhaustion without compromising regulatory controls.

Gout is a prevalent and incapacitating disease triggered by the deposition of monosodium urate (MSU) crystals in joints, which are also massively infiltrated by neutrophils. The interaction of the latter with MSU crystals triggers several responses, including the generation of inflammatory mediators and of neutrophil extracellular traps (NETs). Though some of the signaling events mobilized by MSU in neutrophils have been described (e.g., Src family kinases, Syk, PKC, PI3K), the picture remains fragmentary. Likewise, the impact of these signaling events on cellular responses is incompletely understood. In this study, we examined transcriptomic changes triggered by MSU in neutrophils and their impact on the corresponding proteins, as well as the role of various signaling pathways in prominent functional responses. We report for the first time that neutrophils can secrete the monocyte chemoattractant, CCL4, in response to MSU. Accordingly, we found that transcription factors NF-κB, CREB, and C/EBP are belatedly activated by MSU crystals, and at least the former is involved in chemokine generation. Moreover, we show that MAPKs and Akt are activated by MSU in neutrophils, that they are under the control of TAK1 and Syk, and that they participate in cytokine generation and NETosis. In the latter instance, we found the phenomenon to be independent of endogenous ROS, but under the control of PAD4. We finally provide evidence that endogenous factors contribute to the belated phosphorylation of kinases and transcription factors in response to MSU. Collectively, our findings unveil potentially important therapeutic targets for gouty arthritis., (Copyright © 2020 Tatsiy, Mayer, de Carvalho Oliveira, Sylvain-Prévost, Isabel, Dubois and McDonald.)

Mast cell (MC) activation via aggregation of the high affinity IgE receptor (FcεRI) causes degranulation and release of proinflammatory mediators in a process that involves the reorganization of the actin cytoskeleton. However, the regulatory pathways and the molecular links between cytoskeletal changes and MC function are incompletely understood. In this study, we provide genetic evidence for a critical role of the actin-regulatory proteins Coronin1a (Coro1a) and Coro1b on exocytic pathways in MCs: Coro1a(-/-) bone marrow-derived MCs exhibit increased FcεRI-mediated degranulation of secretory lysosomes but significantly reduced secretion of cytokines. Hyperdegranulation of Coro1a(-/-) MCs is further augmented by the additional loss of Coro1b. In vivo, Coro1a(-/-)Coro1b(-/-) mice displayed enhanced passive cutaneous anaphylaxis. Functional reconstitution assays revealed that the inhibitory effect of Coro1a on MC degranulation strictly correlates with cortical localization of Coro1a, requires its filamentous actin-binding activity, and is regulated by phosphorylation of Ser2 of Coro1a. Thus, coronin proteins, and in turn the actin cytoskeleton, exhibit a functional dichotomy as differential regulators of degranulation versus cytokine secretion in MC biology., (© 2011 Föger et al.)

Since cytokines are potent immunoregulators that can determine the fate of an immune response, their expression is tightly regulated at the transcriptional level. Recent research, however, has also revealed complex post-translational mechanisms through which cytokine secretion, and thereby cytokine activity, is regulated. Here, we review the progress in our understanding of the portfolio of pathways that regulate cytokine intracellular storage, transport, and release. Like other secreted proteins, cytokines utilize canonical and non-canonical secretory pathways for extracellular release. Illustrated by IL-1β, IL-2, TNF-α, IL-12 and IL-15 secretion as selected examples, we discuss common and alternative cytokine secretion pathways and relate them to the consequences these distinct pathways have for cytokine function, mode of action and stability., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Human monocytes respond to a variety of stimuli with a complex spectrum of activities ranging from acute defense mechanisms to cell differentiation or cytokine release. However, the individual intracellular signaling pathways related to these functions are not well understood. CXC chemokine ligand 4 (CXCL4) represents a broad activator of monocytes, which induces acute as well as delayed activities in these cells including cell differentiation, survival, or the release of ROS, and cytokines. Here, we report for the first time that CXCL4-treated monocytes significantly upregulate sphingosine kinase 1 (SphK1) mRNA and that CXCL4 induces SphK1 enzyme activity as well as its translocation to the cell membrane. Furthermore, we could show that pharmacological inhibition of SphK results in reversal of CXCL4-induced monocyte survival, cytokine expression, and release of oxygen radicals, which was confirmed by the use of SphK1-specific siRNA. CXCL4-mediated rescue from apoptosis, which is accompanied by inhibition of caspases, is controlled by SphK1 and its downstream element Erk. Taken together, these data assign SphK1 as a central regulator of acute and delayed monocyte activation and suggest SphK1 as a potential therapeutic target to suppress pro-inflammatory responses induced by CXCL4.

Extracellular ATP mediates a diverse array of biological responses in many cell types and tissues, including immune cells. We have demonstrated that ATP induces purinergic receptor P2X(7) mediated membrane permeabilization, apoptosis, and cytokine expression in murine mast cells (MCs). Here, we report that MCs deficient in the expression of the P2X(7) receptor are resistant to the ATP-induced membrane permeabilization and apoptosis. However, ATP affects the tyrosine phosphorylation pattern of P2X(7)knockout cells, leading to the activation of ERK1/2. Furthermore, ATP induces expression of several cytokines and chemokines in these cells, including IL-4, IL-6, IFN-gamma, TNF-alpha, RANTES, and MIP-2, at the mRNA level. In addition, the release of IL-6 and IL-13 to cell-conditioned medium was confirmed by ELISA. The ligand selectivity and pharmacological profile indicate the involvement of two P2X family receptors, P2X(1) and P2X(3). Thus, depending on genetic background, particular tissue microenvironment, and ATP concentration, MCs can presumably engage different P2X receptor subtypes, which may result in functionally distinct biological responses to extracellular nucleotides. This finding highlights a novel level of complexity in the sophisticated biology of MCs and may facilitate the development of new therapeutic approaches to modulate MC activities.

Platelet factor 4 (PF4; CXCL4) is an abundant platelet alpha-granule CXC chemokine with unique functions. Although lacking a chemotactic activity, PF4 initiates a signal transduction cascade in human monocytes leading to the induction of a broad spectrum of acute and delayed functions including phagocytosis, respiratory burst, survival, and the secretion of cytokines. Surprisingly, although these monocyte functions are well defined, only very limited information exists on the specific signaling pathways that are involved in the regulation of these biological responses. By using specific inhibitors and direct phosphorylation/activation studies, we show in the present study that PF4-mediated respiratory burst is dependent on a very rapid activation of PI3K, Syk, and p38 MAPK. Moreover, monocyte survival and differentiation instead is controlled by a delayed activation of Erk, with an activity peak after 6 h of stimulation. The inhibition of Erk completely reverted PF4-mediated protection against apoptosis. Finally, even though JNK is rapidly activated in PF4-treated monocytes, it is dispensable for the regulation of survival and respiratory burst. However, PF4-induced up-regulation of chemokine and cytokine mRNA and protein requires a sustained activation of JNK and Erk. Taken together, PF4-stimulated immediate monocyte functions (oxygen radical formation) are regulated by p38 MAPK, Syk, and PI3K, whereas delayed functions (survival and cytokine expression) are controlled by Erk and JNK.

Extracellular ATP and other nucleotides act through specific cell surface receptors and regulate a wide variety of cellular responses in many cell types and tissues. In this study, we demonstrate that murine mast cells express several P2Y and P2X receptor subtypes including P2X(7), and describe functional responses of these cells to extracellular ATP. Stimulation of bone marrow-derived mast cells (BMMC), as well as MC/9 and P815 mast cell lines with millimolar concentrations of ATP, resulted in Ca(2+) influx across the cellular membrane and cell permeabilization. Moreover, brief exposures to ATP were sufficient to induce apoptosis in BMMCs, MC/9, and P815 cells which involved activation of caspase-3 and -8. However, in the time period between commitment to apoptosis and actual cell death, ATP triggered rapid but transient phosphorylation of multiple signaling molecules in BMMCs and MC/9 cells, including ERK, Jak2, and STAT6. In addition, ATP stimulation enhanced the expression of several proinflammatory cytokines, such as IL-4, IL-6, IL-13, and TNF-alpha. The effects of ATP were mimicked by submillimolar concentrations of 3-O-(4'-benzoyl)-benzoyl-benzoyl-ATP, and were inhibited by pretreatment of mast cells with a selective blocker of human and mouse P2X(7) receptor, 1[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine, as well as oxidized ATP. The nucleotide selectivity and pharmacological profile data support the role for P2X(7) receptor as the mediator of the ATP-induced responses. Given the importance of mast cells in diverse pathological conditions, the ability of extracellular ATP to induce the P2X(7)-mediated apoptosis in these cells may facilitate the development of new strategies to modulate mast cell activities.

Following the identification of the interleukin (IL)-1 type II receptor as a prototypic decoy receptor, nonsignaling receptors with decoy functions have been identified for members of the IL-1/IL-18, tumor necrosis factor, IL-10, and IL-13 receptor families. Moreover, the silent receptor D6 is a promiscuous decoy and scavenger receptor of inflammatory chemokines. The type II IL-1 decoy receptor also acts as a dominant-negative molecule. Intracellular pathways of inhibition of IL-1 and Toll-like receptor (TLR) signaling have been identified. In particular, recent results suggest that the Toll/IL-1 receptor (TIR) family member TIR8, also known as single immunoglobulin IL-1-related receptor (SIGIRR), is a negative regulator of IL-1 and TLR signaling. Thus, extracellular and intracellular decoys tune the activation of members of the IL-1/TLR receptor family.

Although many membrane components have been described to be involved in the activation of cells by bacterial lipopolysaccharide (LPS), the question remains whether LPS, once internalized by target cells, is also capable of interacting with cytoplasmic elements in such a way that activation of cells results independently of receptor engagement. This is an important aspect to consider with respect to the development of strategies aimed at attenuating adverse effects of LPS in the framework of bacterial infections. In this study, human monocyte derived macrophages as representatives of one of the primary target cells activated by LPS, were microinjected with LPS to circumvent exogenous LPS stimulation. Parameters correlating to cytoplasmic activation of the nuclear transcription factor NFkappaB (intracellular calcium mobilization), to nuclear translocation of the NFkappaB p65 subunit and to mRNA-transcription of inflammatory cytokines known to be expressed upon exogenous LPS-stimulation and to require NFkappaB activation (interleukin-1beta, interleukin-6, tumor necrosis factor alpha) were investigated. In addition, the LPS-reporter cell line 3E10, which contains a reporter gene under the control of an NFkappaB-inducible promoter was analyzed with respect to NFkappaB nuclear translocation and reporter gene expression. None of the cellular systems used and none of the parameters investigated led to the observation that intracellular LPS leads to activation of the cells in comparison to external LPS stimulation. These experiments allow the conclusion that LPS in the cytoplasmic compartment does not lead to NFkappaB translocation, cytokine mRNA transcription, and NFkappaB dependent protein expression and suggest that these activation parameters require the interaction of LPS with external membrane components., (Copyright 2002 Wiley-Liss, Inc.)

Platelet factor 4 (PF-4), a platelet-derived CXC chemokine, has been shown to induce the differentiation of monocytes into a subset of macrophages that lack the expression of HLA-DR Ag. This suggests a potential role for PF-4 in the modulation of monocyte-dependent T cell activation. Using an Ag-specific stimulation model in which T cells were cocultured with monocytes in the presence of recall Ags, we could show that under these conditions PF-4-treatment caused a strong decrease of T cell proliferation as well as of IFN-gamma release. However, inhibition of T cell functions such as proliferation, IL-2 release, and IL-2 mRNA production did also occur when isolated T cells were activated in the absence of monocytes with immobilized Abs directed against CD3 in combination with cross-linked anti-CD28 Abs. The effect could be reversed when low concentrations of exogenous IL-2 instead of anti-CD28 were used as a costimulus in combination with anti-CD3 Abs. Further evidence for direct modulation of T cell function by PF-4 was obtained by the detection of specific binding sites for the chemokine on the surface of these cells. Taken together, our results show that specific binding of PF-4, resulting in the down-regulation of the IL-2-release correlates with the inhibition of functions in activated T cells.

Lipopolysaccharide (LPS), also known as endotoxin, is a compound of the cell wall of Gram-negative bacteria, which has been demonstrated to induce inflammatory reactions in vitro as well as in vivo, including lethal shock. A great number of different cells have been documented to be reactive to LPS, e.g. monocytes/macrophages, vascular cells, polymorphonuclear cells, and even B lymphocytes. We have now established that T lymphocytes could also contribute to an inflammatory reaction to LPS. LPS is a potent inducer of human T-lymphocyte proliferation and cytokine production. The activation of T lymphocytes by LPS requires direct cell-to-cell contact with viable accessory monocytes. This interaction was found to be MHC-unrestricted, but strongly dependent on costimulatory signals provided by B7/CD28 interactions. The frequency of responding T lymphocytes is less than 1:1000. A very exciting finding was that not only monocytes, but also CD34+ hematopoietic stem cells, which circulate in peripheral blood in very low frequency, exert essential accessory cell activity during stimulation of T lymphocytes by LPS. In contrast, the response of T lymphocytes to conventional recall antigens is not controlled by blood stem cells. These conclusions are based on the observation that depletion of CD34-positive blood stem cells resulted in a complete loss of LPS-induced T-lymphocyte stimulation. Addition of CD34-enriched blood stem cells led to a recovery of reactivity of T lymphocyte to LPS. The characteristics of T-lymphocyte activation indicate that LPS is neither active as a mitogen, or as a superantigen, or as a classical antigen, but may activate T lymphocyte through a new, so far undescribed, mechanism. Furthermore, the involvement of hematopoietic blood stem cells in the activation of T lymphocytes by LPS demonstrates a role of these cells in inflammatory and immunological events.

The present study was designed to investigate the effect of a series of cytokines on the release of the type II IL-1 decoy receptor, which represents a unique pathway of negative regulation of the IL-1 system. After 20 min, IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IFN-gamma, granulocyte-CSF, macrophage-CSF, and TGF-beta had little or no effect on IL-1 binding by human polymorphonuclear cells. In contrast granulocyte-macrophage-CSF and, to a greater extent, TNF markedly reduced IL-1 binding. The action of TNF was rapid, reaching 50% of its maximum (80%) at 2 min, and plateauing at 20 min with decrease in receptor number and no significant change in affinity. Loss of surface receptor was associated to rapid release of a 45-kDa IL-1-binding molecule identified as the decoy RII. TNF-induced release of the decoy RII was independent of protein synthesis and reactive oxygen intermediates. Monocytes showed a similar response to TNF, except for the size of the released molecule (approximately 60 kDa). TNF induced rapid release of its own receptors. In contrast IL-1beta affected neither its own receptors nor the TNF-R. TNF and, more efficiently, PMA caused release of the decoy RII in fibroblasts transfected with the full-length decoy RII or with a cytoplasmatic deletion mutant. TNF-induced decoy RII release represents an unidirectional pathway of communication in the interplay between the IL-1 and TNF system.

Under certain conditions, natural killer (NK) cells accumulate rapidly at extrahematic sites. In an effort to define the mechanisms underlying the recruitment of NK cells in tissues, we investigated their ability to adhere and transmigrate across endothelial cell (EC) monolayers. A considerable proportion of NK cells adhered to EC and about 30-40% of the adherent NK cells transmigrated across EC. NK cells were 2-3 times more efficient than resting T cells. Exposure of NK cells to IL-2 and IL-12 augmented their adhesive ability, while IL-4 had an inhibitory effect. mAb directed against CD18 and CD11a inhibited binding and migration of NK cells across resting or IL-1-activated EC, whereas anti-CD11b and Cd11c did not. Using IL-1-activated EC, it was found that anti-VLA-4 and anti-VCAM-1 mAb utilized in concert with anti-CD18 significantly reduced adhesion and transmigration. The CS-1 peptide of fibronectin (which recognizes VLA-4), when used in concert with anti-CD18 and anti-VCAM-1 (but not anti-VLA-4), caused a small, but significant increase in inhibition. Thus, LFA-1 and VLA-4 are crucial determinants of the adhesive and migratory interaction with the vascular endothelium.

The beneficial effects of the E. coli extract Colibiogen inj. N (Cb) observed in therapy of inflammatory bowel diseases, allergies, or gastrointestinal tumors are possibly mediated by the induction of cytokines in human leukocytes or vascular cells. Thus, the induction of the cytokines interleukin 1 (IL1), IL6 and tumor necrosis factor (TNF) in human mononuclear cells (MNC) and vascular cells was investigated in vitro. Various administration forms of the extract (including Cb-inj. N, Cb-oral, and Cb-infantibus N) induced the release of IL1 and IL6 from MNC. The compounds stimulated TNF production less potently, possibly due to a lower sensitivity of the TNF assay system, as compared to the IL1 and IL6 detection system. The MNC produced the cytokines with a kinetics similar to that observed with other stimuli. Monospecific antibodies abolished the respective cytokine activity in the biological assays. Addition of submaximal amounts of endotoxin potently enhanced the IL1- and IL6-inducing activity of the bacterial extract, indicating synergism of the extract and endotoxin. These results provide evidence that cytokines produced by MNC following administration of the tested bacterial extract may contribute to the regulation of the immune response during therapy of gastrointestinal tumors. At present the in vivo production of cytokines following treatment with the bacterial extract tested is under investigation in a phase III study.

Cytokine gene expression was investigated in the germinal centre constituents of follicular dendritic cells and germinal centre T cells and compared to the mRNA expression of nongerminal centre tonsillar cells. Cells were isolated from human tonsils by preenrichment with MACS and subsequent FACS sorting. Cytokine gene expression was investigated by intronspanning RT-PCR for IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, IFN-alpha, IFN-beta, IFN-gamma, and TNF-alpha. Frequency of cytokine-producing cells and the cytokine production pattern of single cells were determined by single cell PCR. Furthermore, cytokine protein expression was investigated by immunohistology. Using these methods, we found a strong production of IL-1 beta mRNA and protein in a small percentage of FDC. Germinal centre T cells showed production of IL-2, IL-4, IL-10, IFN-gamma, and TNF-alpha mRNA. The mRNAs of these cytokines could also be detected at the single cell level; as they were produced in a high percentage of germinal centre T cells, whereas immunohistological staining was negative, we conclude that a high percentage of germinal centre T lymphocytes produce IL-2, IL-4, IL-10, IFN-gamma, and TNF-alpha in low concentrations in contrast to T cells outside the germinal centre, in which strong cytokine production in few cells was shown earlier.

Different species of mycobacteria differ in their capacity to induce the production of tumor necrosis factor-alpha (TNF-alpha) by human monocytes in vitro. Whereas M. tuberculosis is a potent inducer of TNF-alpha, M. leprae is much less potent. TNF-alpha production is found to be associated with the availability of H2O2 generated by activated monocytes, as superoxide enhancing H2O2 concentration increases and catalase degrading H2O2 decreases TNF-alpha production. Furthermore, M. kansasii with high intrinsic catalase induce less TNF-alpha than mycobacteria with low intrinsic catalase. In vitro infection of monocytes with M. tuberculosis leads to an impairment of the antigen-presenting capacity, as determined by a reduction of antigen-induced T cell proliferation and interferon gamma (IFN-gamma) production. Of crucial importance is this impairment is the M. tuberculosis-induced down-modulation of MHC class II antigens. The role of TNF-alpha in vivo is reflected in patients with various forms of leprosy. In skin lesions of lepromatous leprosy patients TNF-alpha, interleukin 1 beta (IL-1 beta), and INF-gamma production are found to be rare, whereas these cytokines are well expressed in skin lesions of patients with tuberculoid leprosy. After multidrug chemotherapy an increase of local cytokine production is found. Taken together, these findings suggest that components of mycobacteria may interfere with local cell-mediated immune reactions in vivo. The molecular mechanisms involved in these local responses need to be defined.

Skin biopsies from untreated and MDT-treated patients were examined for infiltrating cells and cells producing the cytokines TNF-alpha, IFN-gamma, and IL-1 beta using immunohistochemistry. Biopsy specimens from untreated tuberculoid leprosy patients were characterized by the presence of cells producing TNF-alpha, IFN-gamma, and IL-1 beta and of subepidermal Langerhans cells. These cells were rarely found or completely absent in biopsies of untreated lepromatous leprosy patients, but tended to increase under MDT. In a short-term therapy trial for three months with brodimoprim, dapsone, and rifampicin, 12 patients were monitored by follow-up biopsies. Semiquantitative PCR for mycobacterial DNA revealed two groups of patients: one group in which mycobacterial DNA in follow-up biopsies remained constant and a second group in which a decrease of mycobacterial DNA during therapy was noted. Immunophenotyping in these follow-up biopsies revealed that in the latter group IFN-gamma-positive cells and Langerhans cells were present and gamma delta T cell receptor-positive cells tended to decrease during therapy. In contrast, in patients whose mycobacterial DNA did not change during therapy, these phenotypical manifestations were not observed. We therefore, conclude that assessment of mycobacterial DNA in combination with phenotyping of infiltrating cells and determination of cytokine patterns may be useful tools in establishing criteria for the effectiveness and duration of MDT in patients with leprosy.

The aim of this study was to determine cytokines in human leprosy lesions by means of immunohistologic examination. Cryostat sections of skin biopsies from 57 patients with various forms of leprosy were immunostained according to the APAAP method, using monoclonal antibodies against interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and, in addition, against CD 1 antigen. Granulomas in biopsies of untreated patients with tuberculoid leprosy showed large amounts of cells positive for IL-1 beta, TNF-alpha, IFN-gamma, and CD 1, whereas no positive signals could be detected in untreated patients with lepromatous leprosy. However, in those biopsies obtained from lepromatous leprosy patients undergoing chemotherapy, positive staining for cytokines as well as subepidermal Langerhans cells increased to a detectable amount. Remarkably, in tuberculoid leprosy patients, the number of IL-1 beta--positive cells did not vary under therapy, while the number of TNF-alpha and IFN-gamma reactive cells decreased. These results suggest that immunohistologic determination of cytokines in combination with the assessment of subepidermal Langerhans cells in human leprosy lesions may be used as a parameter for the patient's status of cell-mediated immunity under chemotherapeutic treatment.