IL-18BPa:Fc cooperates with immunosuppressive drugs in human whole blood

The proinflammatory cytokine interleukin (IL)-18 appears to be involved in the pathogenesis of diseases associated with immunoactivation and inflammation. Consequently, blockage of IL-18 bioactivity by use of IL-18 binding protein (IL-18 BP) is likely a promising therapeutic concept. In the present study, we investigated immunomodulatory activities of IL-18 BPa:Fc in human whole blood cultures. We report that IL-18 BPa:Fc (200 ng/mL) significantly inhibited lipopolysaccharide (LPS, 10 ng/mL)/IL-12 (5 ng/mL)-induced release of interferon-gamma (IFNgamma) and matrix metalloproteinase-9 (MMP-9) from whole blood cultures of healthy donors. Notably, IL-18 BPa:Fc (200 ng/mL) further reinforced dexamethasone (5 nM)- or mycophenolic acid (2 microM)-mediated reduction of LPS/IL-12-induced IFNgamma production by an additional 50.5 or 49.9%, respectively. To investigate effects of IL-18 BP:Fc in the context of autoimmune diseases, experiments were performed with whole blood obtained from patients with systemic lupus erythematosus or Wegener's granulomatosis undergoing immunosuppressive therapy. After ex vivo stimulation with LPS (10 ng/mL), production of IFNgamma and MMP-9 was determined. Both mediators likely contribute to renal inflammation frequently seen in these diseases. In accord with the aforementioned data, LPS (10 ng/mL)-induced IFNgamma was significantly reduced by coincubation with IL-18 BPa:Fc at 200 ng/mL. IL-18 BPa:Fc also inhibited production of MMP-9. The present data demonstrate that IL-18 BPa:Fc has the potential to amplify anti-inflammatory actions of immunosuppressive drugs, and thus may prove to be a valuable novel pharmacological component in the treatment of human autoimmune diseases.     

Regulatory mechanisms of IL-2 and IFNgamma suppression by quercetin in T helper cells

Quercetin is a popular flavonoid compound that is biosynthesized by plants; it is suggested to modulate a variety of inflammatory responses of macrophages and T lymphocytes. Oral administration of quercetin in arthritic rats dramatically diminishes clinical signs of arthritis. Moreover, quercetin ameliorates experimental autoimmune encephalomyelitis, which is associated with Th1-mediated immune responses. Like quercetin inhibits macrophage-induced cytokine production, it also blocks IL-12-dependent JAK-STAT signaling in Th cells. Despite the anti-inflammatory effects of quercetin acting through Th cells, the regulatory mechanisms remain unclear. Here, we studied the function of quercetin in Th cells and found that quercetin suppressed both IFNgamma and IL-2 production upon T cell receptor stimulation. Furthermore, we uncovered the regulatory mechanisms of quercetin involved in the inhibition of cytokine production during Th cell activation. The fact that quercetin-derived IFNgamma suppression was blocked in T-bet-deficient Th cells demonstrated quercetin act through the modulation of T-bet expression. Whereas IL-2 inhibition by quercetin was independent of T-bet expression, quercetin diminished IL-2R alpha expression, which is critical for positive regulatory loop of IL-2 autoactivation. Taken together, quercetin is suggested to repress both IFNgamma and IL-2 cytokine production by independent mechanisms; T-bet-dependent IFNgamma suppression and IL-2R alpha-dependent IL-2 inhibition.     

Interferon gamma-producing ability in blood lymphocytes of patients with lung cancer through activation of the innate immune system by BCG cell wall skeleton

An in vitro assay system was developed to assess the potency of the human innate immune system by measurement of IL-12, IL-18, IL-10 and IFNgamma in the supernatants of bacillus Calmette-Guerin cell wall skeleton (BCG-CWS)-stimulated blood samples. BCG-CWS is a ligand for Toll-like receptor (TLR) 2 and 4, and activates monocytes to macrophages (Mphi), and immature dendritic cells to mature antigen-presenting cells (APC). This system was found to allow the discrimination of immune suppressive states in patients with lung cancer from normal immune states in light of the cytokine profile. The following results were deduced from analyses of BCG-CWS-stimulated blood samples of lung cancer patients with reference to normal subjects. (1) The levels of production of IFNgamma and IL-10 by lymphocytes were decreased. (2) IL-12 p40 production by monocytes/Mphi was upregulated, while that of IL-10 was downregulated. (3) IL-18 was detected in all patients in a range similar to normal subjects. (4) Responses of lymphocytes to IL-2 and IL- 18 in terms of IFNgamma production were diminished. (5) The upregulated IL-12 levels were recovered to within the normal range in most patients after tumor resection. (6) Male patients showed more severe suppression of IL-12/IL-18-mediated IFNgamma production than female patients. Thus, the lesser IFNgamma production observed in patients' blood with high IL-12 p40 levels in response to BCG-CWS may reflect the production of p40 dimers or IL-23 instead of p70, or the presence of some unknown pathways to prohibit the interface between the innate and acquired immune systems. BCG-CWS-mediated Toll signaling may participate in IFNgamma induction for lymphocytes through Mphi/APC IL-12/I-18 modulation.     

Effects of serotonin and serotonergic agonists and antagonists on the production of interferon-gamma and interleukin-10.

Serotonin (5-HT) is a neurotransmitter and an immune modulator. In vitro, antidepressants with a serotonergic mode of action have, at concentrations within the therapeutical range, negative immunoregulatory effects, i.e., they increase the production rate of interleukin-10 (IL-10), a negative immunoregulatory cytokine. We have hypothesized that part of these effects may be explained by the serotonergic activities of antidepressants on immunocytes. This study was carried out to examine the effects of 5-HT, p-chlorophenylalanine (PCPA), a 5-HT depleting agent, flesinoxan (a 5-HT1A agonist), m-chlorophenylpiperazine (mCPP; a 5-HT2A/2C agonist), and ritanserin (a 5-HT2A/2C antagonist) on the production rate of interferon-gamma (IFNgamma), a proinflammatory cytokine, and IL-10 by whole blood stimulated with polyclonal activators. The IFNgamma/IL-10 production ratio was computed, since this ratio reflects the pro- versus anti-inflammatory capacity of cultured whole blood. We found that: 1) 5-HT, 150 ng/mL, 1.5 microg/mL, and 15 microg/mL significantly decreased the IFNgamma/IL-10 ratio; 2) PCPA (5 microM) significantly suppressed the production of IFNgamma and IL-10; 3) flesinoxan (15 ng/mL; 1.5 microg/mL) had no significant effects on the production of the above cytokines; and 4) mCPP (2.7 microg/mL) and ritanserin (5.0 microg/mL) suppressed the IFNgamma/IL-10 ratio. It is concluded that intracellular 5-HT may be necessary for an optimal synthesis of IFNgamma and IL-10, and that extracellular 5-HT concentrations at or above serum values may suppress the production of the proinflammatory cytokine IFNgamma. The negative immunoregulatory effects of antidepressive drugs are probably not related to their serotonergic activities.     

Harnessing CD36 to rein in inflammation

Maintaining health requires a dynamic balance between the influence of pro-inflammatory and anti-inflammatory mediators. While inflammation serves an important protective role against infection, unrestrained inflammation is acutely lethal and unresolved inflammation contributes to a broad range of chronic disorders. Immunotherapy with cytokines themselves or cytokine antagonists faces strict limitations due to efficacy, safety and cost. More successful treatment of the pro-inflammatory component of chronic disorders may emerge from strategies designed to reset the balance between pro and anti-inflammatory cytokines through physiological regulatory pathways. One emerging avenue for this approach is exploitation of the link between the cell surface protein CD36 and the anti-inflammatory cytokine interleukin-10 (IL-10). Agents that increase CD36 expression and agents that directly bind to CD36 have anti-inflammatory properties that may directly relate to induction of IL-10. The immunosuppressive effects of apoptotic cells were first reported more than a decade ago and have since been tested in animal models and several clinical trials. A recent publication demonstrates that induction of IL-10 by apoptotic cells is largely dependent upon the interaction between apoptotic cells and CD36, the receptor on monocytes and macrophages for apoptotic cells. This provides a direct mechanistic link between CD36 engagement and IL-10 induction, opening up new possibilities for using CD36 ligands, agents that increase CD36 expression or a combination of both to modulate inflammation and treat, or even prevent, an important set of chronic disorders.     

Gender-related differences in subacute fumonisin B1 hepatotoxicity in BALB/c mice

Fumonisin B1 (FB1), a potent mycotoxin prevalent in corn, is a carcinogen and causative agent of various animal diseases. Species and sex variations to chronic FB1 toxicity have been reported. Free sphingoid bases and cytokine levels are the two major biochemical alterations of FB1 in vivo and may explain any sex differences in FB1 toxicity. Male and female BALB/c mice (5/group) were injected subcutaneously with either saline vehicle or 2.25 mg/kg/day of FB1 for 5 days. One day after the last injection females showed a greater increase in circulating alanine aminotransferase and greater number of apoptotic cells in liver after FB1 treatment than males, indicating greater hepatotoxicity. Peripheral leukocytic counts, including neutrophils, were increased in females only after FB1 treatment. The increased toxicity in females correlated with a greater increase of sphinganine and sphingosine levels in liver after FB1 treatment compared to males. No sex differences in kidney sphinganine or sphingosine levels were observed after FB1 treatment. Previously we have shown the induction of tumor necrosis factor alpha (TNFalpha) in FB1-induced hepatotoxicity. While in males FB1 treatment caused increased expression of TNFalpha, interleukin (IL)-12 p40, interferon gamma (IFNgamma), IL-1beta, IL-6 and IL-10, females showed an increased expression of IL-6 only, and a downward modulation of IFNgamma, indicating gender differences in cytokine pathways in liver activated by FB1. The basal expression of TNFalpha, IL-12 p40, IL-1beta and IFNgamma in liver of females was higher compared to males. Gender differences in alterations of free sphingoid bases and cytokine modulation after FB1 treatment suggest their possible involvement in sex-dependent differential hepatotoxicity in mice.     

IL-18 initiates release of matrix metalloproteinase-9 from peripheral blood mononuclear cells without affecting tissue inhibitor of matrix metalloproteinases-1: suppression by TNFα blockage and modulation by IL-10

The proinflammatory cytokine interleukin (IL)-18 appears to be involved in the etiology of a variety of pathological conditions, among them rheumatoid arthritis and atherosclerosis as well as tumor growth and metastasis. As biological activity of matrix metalloproteinase-9 (MMP-9) has been identified as a hallmark in the pathogenesis of these diseases, effects of IL-18 on MMP-9 production by human peripheral blood mononuclear cells (PBMC) were investigated. Moreover, effects of immunopharmacological intervention by anti-tumor necrosis factor-alpha (TNFalpha) or IL-10 were evaluated. Here we report that IL-18 augmented production of MMP-9 by PBMC. The potency of IL-18 to induce release of MMP-9 from PBMC was comparable with that of TNFalpha. MMP-9 production was dependent on endogenous production of TNFalpha, as detected by use of neutralizing monoclonal antibodies. Whereas IL-18 and TNFalpha induced the protease, MMP-9 release was not mediated by IFNgamma. IL-18 also induced secretion of MMP-9 from human whole blood cultures. Antiinflammatory IL-10 efficiently downregulated release of MMP-9 from unstimulated and IL-18-activated PBMC. In contrast to MMP-9, secretion of tissue inhibitor of metalloproteinases-1 (TIMP-1) was not augmented by IL-18. Addition of IL-10 enhanced release of TIMP-1 from PBMC. The present study broadens the current pattern of IL-18 proinflammatory actions on PBMC, emphasizes the pivotal role of intermediate TNFalpha production in these responses, and relates IL-18 biological functions to the pathological role of MMP-9.     

S100G expression and function in fibroblasts on colitis induction

Supplementation with interleukin (IL)-10, an important anti-inflammatory cytokine, has shown disappointing efficacy for inflammatory bowel diseases (IBD). IL-10 may down-regulate the expression of other anti-inflammatory mediators following colitis induction. We used a colitis model characterized by hapten-protein visualization, which indicates the site of hapten-protein formation after colitis induction for histological and gene expression analyses. Under IL-10 deficiency, following colitis induction inflammatory changes were reduced, and S100G expression was elevated. S100G was expressed in fibroblasts, and S100G expression was down-regulated by IL-10. S100G suppressed the production of monocyte chemotactic protein-1 (MCP-1) through the inhibition of NF-κB activation. Therefore, S100G, also known as Calbindin-D9k, may be an important anti-inflammatory mediator in fibroblasts following colitis induction, and down-regulation of S100G expression might be one reason for the insufficient performance of IL-10 supplementation.     

IL-1 cytokines in cardiovascular disease: diagnostic, prognostic and therapeutic implications

Interleukins (ILs) are key mediators in the chronic vascular inflammatory response underlying several aspects of cardiovascular disease. Due to their powerful pro-inflammatory potential, and the fact that they are highly expressed by almost all cell types actively implicated in atherosclerosis, members of the IL-1 cytokine family were the first to be investigated in the field of vessel wall inflammation. The IL-1 family is comprised of five proteins that share considerable sequence homology: IL-1alpha, IL-1beta, IL-1 receptor antagonist (IL-1Ra), IL-18 (also known as IFNgamma-inducing factor), and the newly discovered ligand of the ST2L receptor, IL-33. Expression of IL-1s and their receptors has been demonstrated in atheromatous tissue, and serum levels of IL-1-cytokines have been correlated with various aspects of cardiovascular disease and their outcome. In vitro studies have confirmed pro-atherogenic properties of IL-1alpha, IL-1beta and IL-18 such as, up-regulation of endothelial adhesion molecules, the activation of macrophages and smooth muscle cell proliferation. In contrast with this, IL-1Ra, a natural antagonist of IL-1, possesses anti-inflammatory properties, mainly through the endogenous inhibition of IL-1 signaling. IL-33 was identified as a functional ligand of the, till recently, orphan receptor, ST2L. IL-33/ST2L signaling has been reported as a mechanically activated, cardioprotective paracrine system triggered by myocardial overload. As the roles of individual members of the IL-1 family are being revealed, novel therapies aimed at the modulation of interleukin function in several aspects of cardiovascular disease, are being proposed. Several approaches have produced promising results. However, none of these approaches has yet been applied in clinical practice.     

Inhibition of in vitro tumor cell proliferation by cytokines induced by combinations of TLR or TLR and TCR agonists

The objective of this study was to learn from in vitro studies how to better utilize Toll-like receptor (TLR) agonists in controlling tumor growth. One of the primary effects of TLR agonists is induction of cytokine and chemokine production. In order to identify combinations of cytokines or chemokines with optimal ability to inhibit in vitro tumor cell proliferation, a panel of 17 recombinant human or mouse cytokines that have minimal effect on primary cell survival, were tested individually or in combinations of 2, 3 or 4 on a panel of human and mouse chemotherapy sensitive and resistant tumor cell lines. A combination of high (>10 ng/ml) levels of IFNgamma with moderate concentrations of TNFalpha>IFNalpha>IL-6=IL-8 was most effective at inhibiting in vitro tumor cell viability and proliferation with minimal effect on primary cells. We also observed that similar cytokine profile could be induced in vitro PBMC culture by using certain combinations of TLR-TLR and TLR-TCR agonists. Thus, concomitant activation of TLR7/8 with TLR4 or TLR 7/8 with T cell receptor (TCR) in PBMC, amongst all possible paired TLR-TLR and TLR-TCR agonist combinations, produced cytokine mix high in IFNgamma, in combination with IFNalpha, IL-6, IL-8, TNFalpha. Such cytokine mix was equal or more effective tumor cell killing and inhibition of tumor cell proliferation than the best rec-cytokine mixture tested. These results suggest that, TLR and/or TCR agonists combinations generate an optimal mixture of cytokines and chemokines competent in regulating in vitro tumor growth, and imply that realizing such "right cytokine induction" in vivo might be more efficacious than that with individual cytokines or TLR agonists induced cytokine mix.     

IL-27 and autoimmune rheumatologic diseases: The good,the bad,and the ugly

The footprint of cytokines is evident in almost every biological process, such as development, as well as the pathogenesis of the different diseases, immune responses to pathogens, etc. These small proteins are categorized into different functional classes; for instance, they can play a pro-inflammatory or anti-inflammatory role in different situations, or they can confer a polarization to the immune system. Interleukin (IL)-27 is a member of the IL-12 family. Antigen-presenting cells are the primary source of IL-27 production, which exerts its effects by bindings to the IL-27 receptor expressed on the surface of target cells. Interaction of IL-27 and IL-27 receptor leads to activation of the JAK-STAT and p38 MAPK signaling pathways. Most studies focused on the inflammatory effects of this cytokine, but gradually anti-inflammatory effects were also revealed for this cytokine, which changed the traditional perception of the function of this cytokine. The functionality of IL-27 in the pathogenesis of rheumatic diseases has been attributed to a double-blade sword. Hence, novel therapeutic approaches have been devised targeting IL-12 family that has been accompanied with promising results. In this review, we focused on the inflammatory and anti-inflammatory properties of IL-27 in different autoimmune rheumatologic diseases and its plausible therapeutic potentials.     

A new target for the treatment of inflammatory bowel disease: Interleukin-37

Interleukin (IL)-37 belongs to the IL-1 cytokine family. It has anti-inflammatory effects on numerous autoimmune diseases such as asthma, psoriasis, inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), multiple sclerosis (MS) and rheumatoid arthritis (RA). Mechanistically, IL-37 plays an anti-inflammatory role by regulating the expression of inflammatory factors in two ways: binding extracellular receptors IL-18R or transferring into the nucleus with Smad3. IBD is a kind of idiopathic intestinal inflammatory disease with unknown etiology and pathogenesis. Recent researches had proved that IL-37 is negatively involved in the pathogenesis and development of IBD. Among various inflammatory diseases, IL-37 has been shown to regulate inflammatory development by acting on various immune cells such as neutrophils, macrophages (Mϕ), dendritic cells (DCs), T cells and intestinal epithelial cells. This review summarizes the biological role of IL-37, and its immunoregulatory effects on the immune cells, especially anti-inflammatory function in both human and experimental models of IBD.     

In vitro effects of an immunostimulating bacterial lysate on human lymphocyte function

MLBL is an oral immunostimulating vaccine consisting of bacterial standardized lysates obtained by mechanical lysis of different strains of Gram-positive and Gram-negative bacteria that can cause acute and chronic infections of the respiratory tract. Previous studies suggested a stimulating effect of MLBL both on humoral and cellular immune responses. In the present study, the in vitro effects of MLBL on human lymphocyte effector functions and its mechanisms of action were evaluated. The results show that the most remarkable effects of MLBL on the immune system are: i) activation of the IL-2 receptor (IL-2Ralpha) on different lymphocyte subsets (B, CD4+ T and CD8+ T cells) involved both in humoral and cellular immune responses; ii) induction of cytokine synthesis (IL-2, IL-10, IL-12, IFNgamma) in the immune competent cells that induce and regulate immune responses; iii) generation of CD4+ and CD8+ effector T cells. Overall, these results suggest that the therapeutic effect of MLBL on acute and recurrent infections of the respiratory tract is related to its ability to activate the responses of different subsets of immune competent cells both for humoral and cellular immunity. Moreover, these effects can be induced either by direct immune cell activation or through the generation and activation of immune effector cells.     

Moclobemide exerts anti-inflammatory effect in lipopolysaccharide-activated primary mixed glial cell culture

An increasing body of evidence indicates that glial activation and neuroinflammation play an important role in the pathogenesis of psychiatric and neurodegenerative diseases. Activated glial cells secrete various cytokines that influence neurotransmission, hypothalamus–pituitary–adrenal axis activity, neuronal plasticity and neurogenesis. It has been suggested that alterations in cytokine networks are involved in the mechanism of action of antidepressant drugs. Until now, only a few studies demonstrated that some tricyclic antidepressants and selective serotonin reuptake inhibitors reduced production of pro-inflammatory cytokines in brain glia cells. We have investigated for the first time whether the antidepressant, moclobemide (a reversible selective inhibitor of monoamine oxidase-A) has an influence on pro-inflammatory cytokines [interleukin (IL)-1β and tumor necrosis factor (TNF)-α] and anti-inflammatory cytokine (IL-10) in primary rat mixed glial cell cultures stimulated by lipopolysaccharide (LPS). Our results showed that moclobemide used in a wide range of concentrations diminished LPS-stimulated IL-1β and TNF-α mRNAs expression in cellular extracts and remarkably reduced the levels of both pro-inflammatory cytokines in culture medium. In opposite to this, the drug had no influence on IL-10 mRNA and slightly reduced IL-10 concentration. Moreover, moclobemide decreased LPS-stimulated translocation of NFκB p65 subunit into cellular nuclei. These results suggest that moclobemide exerts anti-inflammatory effect in the central nervous system because it affects the balance between pro- and anti-inflammatory cytokines (IL-1β, TNF-α/IL-10) in primary mixed glial cell cultures.     

Synthesis and cytokine modulation properties of pyrrolo[2, 3-d]-4-pyrimidone nucleosides

A series of pyrrolo[2,3-d]pyrimidone nucleosides were synthesized and evaluated for their ability to enhance Type 2 cytokines and to suppress Type 1 cytokines in human T cells activated in vitro. Compounds 16b, 16c, 16d, 18c, and 19b induced substantial enhancement of IL-4 (a Type 2 cytokine) levels while three compounds (16b, 16c, and 16f) showed significant suppression of IFNgamma (a Type 1 cytokine) levels. The results revealed a strict structural requirement for the nucleoside-mediated enhancement of IL-4. Modifications of the ribofuranose moiety of the nucleosides either abolished or dramatically reduced the activity. Both the 5'-hydroxy and 5-carboxamidine are crucial for the activity. Of the few nucleoside analogues that demonstrated enhancement on Type 2 cytokine production, 7-(beta-D-ribofuranosyl)pyrrolo[2, 3-d]-4-pyrimidone-5-carboxamidine (16c) showed a dramatic enhancement (>200%) of IL-4 levels and a significant enhancement (36%) of IL-5 levels. Moreover, this compound showed substantial suppression of the Type 1 cytokines, IFNgamma (42%), IL-2 (54%), and TNFalpha (55%). Similarly, compound 16b showed a substantial enhancement of IL-4 (46%) and suppression of IL-2 (35%), IFNgamma (30%), and TNFalpha (26%). To our knowledge, these are the first nucleoside analogues that induce a Type 2 cytokine bias in T cells. The cytokine modulation property of 16c and 16b merits the therapeutic evaluation of these compounds in treating diseases in which immunopathology is associated with polarized Type 1 cytokine responses.     

Cytokine expression profiles during murine contact allergy: T helper 2 cytokines are expressed irrespective of the type of contact allergen

We have investigated the cytokine response pattern following sensitisation (induction) of BALB/c mice with different chemicals (dinitrochlorobenzene, dinitrofluorobenzene, oxazolone, glutaraldehyde, formaldehyde, trimellitic anhydride, croton oil) and elicitation (challenge) of contact allergy in sensitised animals. The results of our investigations showed that different chemicals induced both T helper (Th) 1 cytokines [interleukin (IL) 2, interferon beta (IFNgamma) [corrected] and Th2 cytokines (IL-4, IL-10) at different stages during murine contact allergy. We also confirmed our previous findings that IL-4 and IL-10 release were up-regulated during the challenge phase regardless the contact allergen used, whereas the release of IFNgamma [corrected] did not show a clear preference for being up- or down-regulated. In our hands, the increased expression of Th2 cytokines after challenge exposure to contact allergens appeared as a stable marker of secondary contact allergenic responses. Quantitative differences in the expression of IL-4 were observed between different contact allergens. The present results clearly indicate that skin sensitisers were able to elicit cytokine response patterns, which could not be related to a clear-cut Th1 or Th2 type of cytokine response. Furthermore, dermal application of contact allergens produced different kinetics of cytokine secretion upon induction and challenge. In our hands, the co-expression of Th1 and Th2 type cytokines appeared as a universal consequence of dermal application of contact allergens to responsive mice. Our results indicate that co-expression of Th1 and Th2 cytokines during contact allergy is an important feature of murine contact allergy in responsive mice and that chemicals differ in their potency to induce the expression of these cytokines. Furthermore, the results do not support the view that different chemicals induce Th1 or Th2 cytokines in a mutually exclusive manner depending on their preference for inducing either contact or respiratory allergy. The results are expected to renew the discussion about the usefulness of the Th1/Th2 paradigm in certain areas of immunotoxicology.