Differential Cytokine Profiles upon Comparing Selective versus Classic Glucocorticoid Receptor Modulation in Human Peripheral Blood Mononuclear Cells and Inferior Turbinate Tissue

Background

Glucocorticoid Receptor agonists, particularly classic glucocorticoids, are the mainstay among treatment protocols for various chronic inflammatory disorders, including nasal disease. To steer away from steroid-induced side effects, novel GR modulators exhibiting a more favorable therapeutic profile remain actively sought after. Currently, the impact of 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium chloride a plant-derived selective glucocorticoid receptor modulator named compound A, on cytokine production in ex vivo human immune cells and tissue has scarcely been evaluated.

Methods and Results

The current study aimed to investigate the effect of a classic glucocorticoid versus compound A on cytokine and inflammatory mediator production after stimulation with Staphylococcus aureus–derived enterotoxin B protein in peripheral blood mononuclear cells (PBMCs) as well as in inferior nasal turbinate tissue. To this end, tissue fragments were stimulated with RPMI (negative control) or Staphylococcus aureus–derived enterotoxin B protein for 24 hours, in presence of solvent, or the glucocorticoid methylprednisolone or compound A at various concentrations. Supernatants were measured via multiplex for pro-inflammatory cytokines (IL-1β, TNFα) and T-cell- and subset-related cytokines (IFN-γ, IL-2, IL-5, IL-6, IL-10, and IL-17). In concordance with the previously described stimulatory role of superantigens in the development of nasal polyposis, a 24h Staphylococcus aureus–derived enterotoxin B protein stimulation induced a significant increase of IL-2, IL-1β, TNF-α, and IL-17 in PBMCs and in inferior turbinates and of IL-5 and IFN-γ in PBMCs.

Conclusion

Notwithstanding some differences in amplitude, the overall cytokine responses to methylprednisolone and compound A were relatively similar, pointing to a conserved and common mechanism in cytokine transrepression and anti-inflammatory actions of these GR modulators. Furthermore, these results provide evidence that selective glucocorticoid receptor modulator-mediated manipulation of the glucocorticoid receptor in human tissues, supports its anti-inflammatory potential.     

Differential induction of IL-1β and IL-6 production by the nontoxic lipid A from Porphyromonas gingivalis in comparison with synthetic Escherichia coli lipid A in human peripheral blood mononuclear cells

Abstract Porphyromonas gingivalis 381 lipid A possesses 1-phospho β(1–6)-linked glucosamine disaccharide with 3-hydroxy-15-methylhexadecanoyl and 3-hexadecanoyloxy-15-methylhexadecanoyl groups at the 2- and 2′-positions, respectively. P. gingivalis lipid A indicated lower activities in inducing interleukin-1β (IL-1β) mRNA expression, pro-IL-1β protein synthesis and IL-1β production than those of synthetic Escherichia coli lipid A (compound 506) in human peripheral blood mononuclear cells (PBMC). The induction of IL-6 mRNA and IL-6 synthesis by P. gingivalis lipid A were comparable to those of compound 506. Herbimycin A, H-7 and H-8, inhibitors of tyrosine kinase, protein kinase C and cyclic nucleotide-dependent protein kinase, inhibited P. gingivalis lipid A- and compound 506-induced IL-1β and IL-6 synthesis. W-7, an inhibitor of calmodulin (CaM) kinase, inhibited only P. gingivalis lipid A-induced IL-1β production. The result suggests that the CaM kinase-dependent cascade is involved in the down-regulation of IL-1β production by P. gingivalis lipid A. P. gingivalis lipid A and compound 506 also functioned in the induction of tyrosine and serine/threonine phosphorylation of several proteins in PBMC. P. gingivalis lipid A inhibited specific binding of fluorescein-labelled E. coli LPS to the PBMC. The nontoxic lipid A of P. gingivalis , having a chemical structure different from toxic compound 506, appears to induce the up- and down-regulation of the differential cytokine-producing activities following the activation of various intracellular enzymes including the CaM kinase through the common receptor sites of LPS.     

The effect of compound nutrients on stress-induced changes in serum IL-2, IL-6 and TNF-alpha levels in rats

The effect of compound nutrients on serum concentrations of the cytokines, such as interleukin (IL)-2, tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 in immobilization and cold water-immersion stressed rat were investigated. Oral (gavage) administration of compound nutrients was found to attenuate the acute and chronic immobilization and cold water-immersion stress-induced increase in serum IL-6 level and decrease in IL-2 level. Compound nutrients exerted different effects on TNF-alpha level in two different models studied, with reduced serum TNF-alpha level in acute stress, while no significant effect in chronic stress. These results suggested that compound nutrients might be proposed as a possible candidate in the research or therapeutic modulation of stress-related disorders.     

Differential inhibition of inducible T cell cytokine secretion by potent iron chelators

Effector functions and proliferation of T helper (Th) cells are influenced by cytokines in the environment. Th1 cells respond to a synergistic effect of interleukin-12 (IL-12) and interleukin-18 (IL-18) to secrete interferon-gamma (IFN-gamma). In contrast, Th2 cells respond to interleukin-4 (IL-4) to secrete IL-4, interleukin-13 (IL-13), interleukin-5 (IL-5), and interleukin-10 (IL-10). The authors were interested in identifying nonpeptide inhibitors of the Th1 response selective for the IL-12/IL-18-mediated secretion of IFN-gamma while leaving the IL-4-mediated Th2 cytokine secretion relatively intact. The authors established a screening protocol using human peripheral blood mononuclear cells (PBMCs) and identified the hydrazino anthranilate compound 1 as a potent inhibitor of IL-12/IL-18-mediated IFN-gamma secretion from CD3(+) cells with an IC(50) around 200 nM. The inhibitor was specific because it had virtually no effect on IL-4-mediated IL-13 release from the same population of cells. Further work established that compound 1 was a potent intracellular iron chelator that inhibited both IL-12/IL-18- and IL-4-mediated T cell proliferation. Iron chelation affects multiple cellular pathways in T cells. Thus, the IL-12/IL-18-mediated proliferation and IFN-gamma secretion are very sensitive to intracellular iron concentration. However, the IL-4-mediated IL-13 secretion does not correlate with proliferation and is partially resistant to potent iron chelation.     

Identification of an imidazopyridine scaffold to generate potent and selective TYK2 inhibitors that demonstrate activity in an in vivo psoriasis model

Herein we report identification of an imidazopyridine class of potent and selective TYK2 inhibitors, exemplified by prototype 6, through constraint of the rotatable amide bond connecting the pyridine and aryl rings of compound 1. Further optimization led to generation of compound 30 that potently inhibits the TYK2 enzyme and the IL-23 pathway in cells, exhibits selectivity against cellular JAK2 activity, and has good pharmacokinetic properties. In mice, compound 30 demonstrated dose-dependent reduction of IL-17 production in a PK/PD model as well as in an imiquimod-induced psoriasis model. In this efficacy model, the IL-17 decrease was accompanied by a reduction of ear thickness indicating the potential of TYK2 inhibition as a therapeutic approach for psoriasis patients.     

Piperine inhibits cytokine production by human peripheral blood mononuclear cells

Piperine, an amide isolated from Piper species (Piperaceae), has been reported to exhibit central nervous system depression, anti-pyretic and anti-inflammatory activity. Immunomodulatory and anti-tumor activity of piperine has been demonstrated in mouse carcinomas. However, there is little information available concerning the effect of piperine on humans. We evaluated the immunopharmacological activity of this compound in human immune cells. Human peripheral blood mononuclear cells (PBMCs) were exposed to piperine, and cell proliferation was determined by the MTS assay. Piperine significantly inhibited phytohemagglutinin-stimulated human PBMC proliferation after exposure for 72 h. This compound inhibited PBMC activity, with an IC(50) of 100.73 ± 11.16 μg/mL. Production of interleukin-2 (IL-2) and interferon-γ (IFN-γ) was measured using an ELISA assay and RT-PCR. Piperine inhibited IL-2 and IFN-γ production in the PBMCs. RT-PCR data indicated that IL-2 and IFN-γ mRNA expression in PBMCs is suppressed by piperine. This compound significantly inhibited the production of these two cytokines by activated PBMCs in a dose-dependent manner. In conclusion, piperine appears to have potential as an immunomodulatory agent for immune system suppression.     

6-Gingerdione Reduces Apoptotic Conditions in HepG2 Cells and Inhibits Inflammatory Cytokine Gene Expression in Alcoholic Liver Injured Zebrafish Larvae

Antioxidant natural products and their analogs especially phenolic compounds, exhibit diverse biological properties, including anti-inflammatory, antioxidant, and anticancer activities. Ginger which is widely used worldwide for various beneficial effects also contains several phenolic antioxidants, and 6-gingerol is one of the natural products studied extensively. However, the molecular mechanism of synthetically synthesized 6-gingerdione (compound 1 ) from 6-gingerol was not known. In this study, compound 1 and methylated 6-gingerdione (compound 2 ) were obtained semi synthetically from 6-gingerol. Compound 1 and 2 are subjected to SwissADME prediction. Then the protective effect of compound 1 was analyzed in 2 % EtOH induced HepG2 cells and zebrafish larvae. Hydroxyl and nitric oxide scavenging assays reveal that compound 1 showed more antioxidant activity than compound 2 at 50 μM. Moreover, compound 1 exhibited good anti-inflammatory activity via lipoxygenase inhibition and proteinase inhibition. Apoptosis and oxidative stress in HepG2 cells were induced by 2 % EtOH and treated with compound 1 . Compound 1 significantly inhibited the EtOH induced nitric oxide production, apoptosis, and ROS generation in HepG2 cells. Encouraged by the in-vitro antioxidant and anti-inflammatory activities, compound 1 was then investigated for its protective effect in 2 % EtOH induced ALD zebrafish larva. Compound 1 protected the zebrafish larvae from liver injury by suppressing inflammatory (COX-2, TNF-α, and IL-1β) and lipogenic genes (C/EBP-α, SREBP1, and IL-1β) while upregulating the antioxidant gene. Our findings indicate that compound 1 synthesized from 6-gingerol ameliorated liver injury that likely, contributes to its potential antioxidant and anti-inflammatory properties.     

Hexadecylphosphocholine-mediated enhancement of T-cell responses to interleukin 2

The effect of low-dose hexadecylphosphocholine (He-PC) on normal peripheral mononuclear cells (PMNC) was studied. Interferon-gamma (IFN-g) production, interleukin 2 (IL-2) receptor, and HLA-DR antigen expression were investigated, representing typical T-cell activation parameters. In PMNC cultures, He-PC dose-dependently enhanced the production of IFN-g, provided IL-2 had been added exogenously. Without IL-2 He-PC was ineffective. In some cultures, at a concentration of 8 micrograms/ml He-PC stimulated the secretion of IFN-g more than 20-fold compared to untreated controls. Although He-PC by itself lacked mitogenic activity, this compound also stimulated IFN-g production in the presence of suboptimal doses of phytohemagglutinin (PHA). Immunofluorescence studies demonstrated that He-PC also increased IL-2 receptor and HLA-DR antigen expression under these experimental conditions. Taken together, these results indicate that He-PC may possess immunomodulatory activity also in vivo, acting as a costimulator for the IL-2-mediated T-cell activation process.     

Direct regulation of IL-2 by curcumin

Interleukin-2 (IL-2) is a crucial growth factor for both regulatory and effector T cells. Thus, IL-2 plays a critical role in the stimulation and suppression of immune responses. Recently, anti-IL-2 antibodies (Abs) have been shown to possess strong IL-2 modulatory activities by affecting the interaction between IL-2 and IL-2 receptors. In this study, we screened an herbal library to identify a compound that regulates IL-2, which resulted in the identification of curcumin as a direct binder and inhibitor of IL-2. Curcumin is a phytochemical with well-known anti-cancer properties. In this study, curcumin mimicked or altered the binding pattern of anti-IL-2 Abs against IL-2 and remarkably inhibited the interaction of recombinant IL-2 with the IL-2 receptor α, CD25. Interestingly, curcumin neutralized the biological activities of IL-2 both in vitro and in vivo. In this report, we elucidated the unsolved mechanism of the anti-cancer effect of curcumin by identifying IL-2 as a direct molecular target. Curcumin, as a small molecule IL-2 modulator, has the potential to be used to treat IL-2 related pathologic conditions.     

Modulation of lipopolysaccharide-induced production of tumor necrosis factor, interleukin 1, and interleukin 6 by synthetic precursor Ia of lipid A

Abstract Endotoxin (lipopolysaccharide, LPS) induces the production of mediators of inflammation, which exerts pathophysiological effects such as fever or shock in mammals. In the present study we have investigated the modulation of LPS by the synthetic non-active tetraacylated precursor Ia of lipid A (compound 406) in the induction of tumor necrosis factor (TNF), interleukin 1 (IL-1) and interleukin 6 (IL-6) in human peripheral blood mononuclear cells (PBMC) and in human peripheral blood monocytes (PBMo). PBMC stimulated with LPS released TNF in a concentration dependent manner. Release of biologically active TNF, IL-1 and IL-6 was first detectable 4 h after LPS stimulation. Compound 406 alone in all concentrations tested did not induce TNF, IL-1 or IL-6 release, intracellular TNF or IL-1β, or mRNA for TNF or IL-1. Added to PBMC 1 h before LPS compound 406 enhanced or suppressed TNF release and suppressed IL-1 and IL-6 release depending on the ratio of concentrations between stimulator (LPS) and modulator (compound 406). In contrast to LPS stimulation alone TNF, IL-1 and IL-6 release in presence of compound 406 was delayed and first detectable after 6 to 8 h. Compound 406 was able to suppress LPS-induced intracellular TNF and IL-1β in PBMC. Added to PBMo 1 h before LPS it totally inhibited the production of mRNA for TNF and IL-1. When added to PBMC 1 h after LPS, TNF release was suppressed in a concentration-dependent way and release of biologically active TNF, IL-1 and IL-6 could again be detected for the first time after 4 h. Compound 406 was not able to inhibit phorbol 12-myristate 13-acetate (PMA)-induced TNF and IL-1 release in PBMo which suggests that its modulating effect is LPS-specific. This study provides evidence that the modulating effect of compound 406 on the LPS induction of TNF, IL-, 1 and IL-6 could be due to competitive binding.     

Cooperation between an anti-T cell (anti-CD28) monoclonal antibody and monocyte-produced IL-6 in the induction of T cell responsiveness to IL-2

CD28 is an Ag of 44-kDa Mr that is expressed on the membrane of the majority of human T cells and that is recognized by mAb 9.3. The functional effects of mAb 9.3 on peripheral blood T cells were studied. mAb 9.3 was not mitogenic, unless it was combined with PMA. When CD28 was cross-linked after binding of mAb 9.3 to the T cell by immobilized or soluble anti-mouse IgG, T cells proliferated in response to rIL-2, provided that monocytes were also present. The additional signal required for IL-2 responsiveness after cross-linking of CD28 could also be delivered in cultures of purified T cells by a cellfree monocyte culture supernatant. Expression of IL-2R on about 10% of the T cells was demonstrated by staining with an anti-IL-2R mAb, and was found to be largely restricted to CD4+ cells. The active compound responsible for the helper signal in the monocyte culture supernatant was identified as IL-6 because purified IL-6 (but not IL-1 beta) had similar activity and because an antiserum to IL-6 (but not an antiserum to IL-1 beta) neutralized the activity of the monocyte supernatant and blocked T cell proliferation. An anti-IL-2R antibody also completely inhibited T cell proliferation induced by the combination of mAb 9.3, IL-2, and IL-6. Our results provide evidence that cross-linking of CD28 induces functional IL-2R and that this activity is dependent on a helper signal provided by monocytes, more specifically IL-6. Moreover, our results indicate that IL-6 (previously called B cell stimulatory factor-2) is active on T cells. If a natural ligand for CD28 can be identified, the mechanism of induction of IL-2 responsiveness described here might explain how T cells become nonspecifically involved in an ongoing cellular immune reaction.     

Novel potent and selective calcium-release-activated calcium (CRAC) channel inhibitors. Part 2: Synthesis and inhibitory activity of aryl-3-trifluoromethylpyrazoles

To identify potent and selective calcium-release-activated calcium (CRAC) channel inhibitors, we examined the structure-activity relationships of the pyrazole and thiophene moieties in compound 4. Compound 25b was found to exhibit highly potent and selective inhibitory activity for CRAC channels and further modifications of the pyrazole and benzoyl moieties of compound 25b produced compound 29. These compounds were potent inhibitors of IL-2 production in vitro and also acted as inhibitors in pharmacological models of diseases resulting from T-lymphocyte activation, after oral administration.     

AMP-activated protein kinase regulates PDGF-BB-stimulated interleukin-6 synthesis in osteoblasts: involvement of mitogen-activated protein kinases

AimWe have previously reported that platelet-derived growth factor (PDGF)-BB stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells, and that the activation of p44/p42 mitogen-activated protein (MAP) kinase, p38MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) is implicated in the IL-6 synthesis. In the present study,we investigated the involvement of AMP-activated protein kinase (AMPK), a regulator of energy metabolism, in the PDGF-BB-stimulated IL-6 synthesis in MC3T3-E1 cells.Main methodsThe levels of IL-6 were measured by ELISA. The phosphorylation of each protein kinases was analyzed by Western blotting. The mRNA levels of IL-6 were determined by real-time RT-PCR.Key findingsPDGF-BB time-dependently induced the phosphorylation of AMPK. Compound C, an inhibitor of AMPK, which reduced PDGF-BB-induced acetyl-CoA carboxylase phosphorylation, dose-dependently suppressed the PDGF-BB-stimulated IL-6 release. In addition, the PDGF-BB-stimulated IL-6 release in human osteoblasts was also inhibited by compound C. The mRNA expression of IL-6 induced by PDGF-BB was markedly reduced by compound C. The PDGF-BB-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase and SAPK/JNK was inhibited by compound C.SignificanceThese results strongly suggest that AMPK positively regulates PDGF-BB-stimulated IL-6 synthesis via the MAP kinases in osteoblasts.     

Modulation of Th1/Th2 cytokines and inflammatory mediators by hydroxychavicol in adjuvant induced arthritic tissues

The present study was undertaken to investigate the anti-arthritic activity of hydroxychavicol (HC) a major phenolic compound isolated from the aqueous extract leaves of plant Piper betle (Piperaceae). The compound showed significant lowering of pro-inflammatory (Th1) cytokine levels in arthritic paw tissue homogenate supernatant viz. IL-2, IFN-γ, and TNF-α with maximum inhibition at higher dose levels of 2 and 4 mg/kg p.o. and enhanced the production of anti-inflammatory (Th2) cytokines IL-4 and IL-5 estimated by cytometric bead array immunoassay. Cytometric bead array uses the sensitivity of amplified fluorescence detection by flowcytometer to measure soluble analytes in a particle based immune assay. This assay can accurately quantitate five cytokines in a 50-μl sample volume. The T-helper (Th1) deviated cells produce detectable level of tumor necrosis factor (TNF-α), interleukin-2 (IL-2), and interferon-gamma (IFN-γ), while the Th2 deviated cells produce significant amount of interleukin-4 (IL-4) and interleukin-5 (IL-5). HC at graded doses also significantly decreased the expression of IL-1β, PGE₂, LTB₄, and nitric oxide levels showing significant inhibition of these parameters. Elevated levels of CD4⁺ T cell specific interferon-gamma (IFN-γ) in splenocytes of arthritic animals was also inhibited in treated animals. The oral LD₀ in both mice and rats was more than 1000 mg/kg.     

Suppressive effect of lipid A partial structures on lipopolysaccharide or lipid A-induced release of interleukin 1 by human monocytes

Abstract Experiments were designed to investigate the significance of lipid A partial structures, precursor Ia (compound 406), and lipid X (compound 401) to serve as antagonists of interleukin 1 (IL-1) release from human mononuclear cells and monocytes induced by lipopolysaccharide (LPS, endotoxin) of Salmonella aborus equi or synthetic Escherichia coli lipid A (compound 506). A definite inhibition mediated by lipid A partial structures on IL-1 release induced by LPS or lipid A was found in repeated experiments. The inhibitory effect was exterted not only on IL-1 release, but also on IL-1 peptide synthesis at the intracellular level. The results also show that lipid A partial structures have suppressive effects even when added 1–4 after LPS or lipid A. We conclude from these results that lipis A partial structures (precursor Ia and lipid X) have potent immunomodulatory effects on LPS- and lipid A-induced IL-1 release and may become useful reagents to study the mechanism of interaction of LPS and lipid A with cells of the immune system.