Immunostimulatory effect by aqueous extract of <Emphasis Type="Italic">Hizikia fusiforme</Emphasis> in RAW 264.7 macrophage and whole spleen cells

Hizikia fusiforme is a commonly used food that possesses potent anti-bacterial, anti-fungal, and anti-inflammatory activities. The immunostimulatory activities of aqueous extract of Hizikia fusiforme (HFAE) in RAW 264.7 macrophages and whole spleen cells were investigated. HFAE activated RAW 264.7 macrophages to produce cytokines such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in a dose-dependent manner. In addition, HFAE induced the mRNA expression of TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophages. Moreover, HFAE stimulated proliferation of whole spleen cells and reference mitogen. Taken together, the results demonstrate that HFAE potently activates the immune function by regulating NO, TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophage and promoting spleen cell proliferation.     

Pb2+ reduces PKCs and NF-κB in vitro

The mechanism of lead (Pb²⁺)-induced neurotoxicity has not yet been fully elucidated. The purpose of this study was to examine the effects of Pb²⁺ on several protein kinase C (PKC) isoforms and the nuclear factor-κB (NF-κB)–I-κB kinase-alpha (IKK-α) axis in cultured neuronal cells. Neurons were isolated from rat fetal brain at the 18th day of gestation of pregnant Sprague Dawley rats and cultured for 10 days before use. Neurons were exposed to Pb²⁺ at concentrations of 10⁻¹⁰, 10⁻⁹, 10⁻⁸, and 10⁻⁷ mol/L for 14 h and antigens of typical PKC-α,β,γ; novel PKC (ε, δ), atypical PKC (λ), NF-κB (p50), and IKK-α were enriched by immunoprecipitation and determined by western blotting. Total, calcium-dependent and independent PKC activities were also determined by counting the transferred γ-³² P in the substrate-histone. The results indicated that inorganic Pb²⁺ significantly reduced all PKC isoforms (α,β,γ, ε, λ) except δ, inhibiting the total, calcium-dependent and calcium-independent PKC activities in a dose-dependent manner. Additionally, Pb²⁺ gradually reduced NF-κB (p50) and IKK-α protein levels. This suggests that Pb²⁺ exhibits varying preference for individual PKC isoforms but reduces the NF-κB–IKK-α axis to a similar extent.     

Interleukin-15 effectively potentiates the in vitro tumor-specific activity and proliferation of peripheral blood γδT cells isolated from glioblastoma patients

γδT cells play a regulatory role in both primary and metastatic tumor growth in humans. The mechanisms responsible for the activation and proliferation of circulating γδT cells should be fully understood prior to their adoptive transfer to cancer patients. We have examined in vitro functional effects of interleukin-15 (IL-15) on highly purified γδT cells isolated from glioblastoma patients. γδT cells constitutively express the heterotrimeric IL-2 receptor (IL-2R) αβγ, but the levels of IL-2Rβ or γ expression were not increased by incubation with saturating amounts of IL-15. IL-15 was shown to induce a maximal γδT cell proliferation, although at much higher concentrations (at least 2000 U/ml) than IL-2 (100 U/ml). Submaximal concentrations of IL-15 plus low concentrations of IL-2 produced an additive proliferative response. In contrast to the IL-2-induced response, this activity was completely or partially abrogated by anti-IL-2Rβ, or anti-IL-2Rγ antibodies, but not by anti-IL-2Rα antibodies. Incubation of γδT cells in the presence of IL-15 resulted not only in the appearance of NK and LAK activity, but also in specific autologous tumor cell killing activity, an additive effect being seen with IL-15 and IL-2. This IL-15-induced tumor-specific activity could be significantly blocked by anti-IL-2Rγ and anti-IL-2R-β mAb, but not by anti-IL-2Rα mAb. Thus, in contrast to IL-2, IL-15 activates tumor-specific γδT cells through the components of IL-2Rβ and IL-2Rγ, but not IL-2Rα. These enhanced in vitro tumor-specific and proliferative responses of γδT cells seen with IL-15 suggest a rational adjuvant imunotherapeutic use of γδT cells in cancer patients. Received: 23 January 1998 / Accepted: 20 May 1998     

Cytokine production by a megakaryocytic cell line

Summary The regulation of megakaryopoeisis by cytokines is not yet well understood. It is possible that autocrine loops are established during megakaryocyte growth and differentiation, aiding in the maturation of these cells. The CHRF-288-11 human megakaryoblastic cell line has been examined for cytokine production in growing cells and cells stimulated to differentiate by the addition of phorbol esters. It has been demonstrated that these cells produce RNA corresponding to the interleukins IL-1α, 1β, 3, 7, 8, and 11, granulocyte-macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), interferon-α (INF-α), and basic fibroblast growth factor (bFGF). Additionaly, RNA corresponding to the receptors for IL-6, GM-CSF, SCF, INF-α,β, bFGF, and monocyte colony stimulating factor (M-CSF) were also expressed by the cells. The receptor for TNF-α was detected immunologically. Analysis at the protein level demonstrated that significant amounts of INF-α, TNF-α, GM-CSF, SCF, IL-1α, and a soluble form of the IL-6 receptor were produced by the cells. Addition of phorbol esters to CHRF-288-11 cells enhances their megakaryocytic phenotype; such treatment also results in increased secretion of INF-α, TNF-α, and GM-CSF. These results suggest that potential autocrine loops are established during the differentiation of CHRF-288-11 cells, which may alter the capability of the cell to differentiate. These findings are similar to those recently obtained for marrow-derived megakaryocytes (Jiang et al.) suggesting that CHRF-288-11 cells provide a useful model system for the study of cytokine release during megakaryocyte differentiation.     

Evidence for the presence of immunoreactive POMC-derived peptides and cytokines in the thymus of the goldfish (Carassius c. auratus)

Summary The presence of immunoreactive pro-opiomelanocortin (POMC)-derived peptides (adrenocorticotropin hormone, β-endorphin, α-melanocyte-stimulating hormone) and of cytokine-like molecules [interleukin (IL)-1α, IL-1β, IL-2, IL-6, tumour necrosis factor-α] was demonstrated in periodic acid-Schiff-positive epithelial cells in the thymus of the goldfish (Carassius c. auratus) using immunocytochemical procedures. POMC-derived peptide- and cytokine-like molecules were localized in the same cell type. Lymphocytes were negative for all the above mentioned molecules. Despite the smaller number of cells positive for neuropeptide- and cytokine-like molecules, our findings suggest that immune-neuroendocrine interactions are likely to occur in the thymus of goldfish.     

Regulation of BCRP (ABCG2) and P-Glycoprotein (ABCB1) by Cytokines in a Model of the Human Blood–Brain Barrier

Brain capillary endothelial cells form the blood–brain barrier (BBB), a highly selective permeability membrane between the blood and the brain. Besides tight junctions that prevent small hydrophilic compounds from passive diffusion into the brain tissue, the endothelial cells express different families of drug efflux transport proteins that limit the amount of substances penetrating the brain. Two prominent efflux transporters are the breast cancer resistance protein and P-glycoprotein (P-gp). During inflammatory reactions, which can be associated with an altered BBB, pro-inflammatory cytokines are present in the systemic circulation. We, therefore, investigated the effect of the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) on the expression and activity of BCRP and P-gp in the human hCMEC/D3 cell line. BCRP mRNA levels were significantly reduced by IL-1β, IL-6 and TNF-α. The strongest BCRP suppression at the protein level was observed after IL-1β treatment. IL-1β, IL-6 and TNF-α also significantly reduced the BCRP activity as assessed by mitoxantrone uptake experiments. P-gp mRNA levels were slightly reduced by IL-6, but significantly increased after TNF-α treatment. TNF-α also increased protein expression of P-gp but the uptake of the P-gp substrate rhodamine 123 was not affected by any of the cytokines. This in vitro study indicates that expression levels and activity of BCRP, and P-gp at the BBB may be altered by acute inflammation, possibly affecting the penetration of their substrates into the brain.     

Targeting the interleukin-15/interleukin-15 receptor system in inflammatory autoimmune diseases

Interleukin (IL)-15 is a dangerous inflammatory cytokine that induces tumor-necrosis factor-α, IL-1β and inflammatory chemokines. It inhibits self-tolerance mediated by IL-2 mediated activation-induced cell death and facilitates maintenance of CD8⁺ memory T-cell survival including that of self-directed memory cells. Disordered IL-15 expression has been reported in patients with an array of inflammatory autoimmune diseases. A series of therapeutic agents that inhibit IL-15 action have been introduced, including the soluble IL-15 receptor (IL-15R) α chain, mutant IL-15, and antibodies directed against the IL-15 cytokine and against the IL-2R/IL-15R β subunit used by IL-2 and IL-15.     

Interleukin-1, tumor necrosis factor-alpha,and transforming growth factor-beta 1 and integrative meniscal repair: influences on meniscal cell proliferation and migration

Introduction  

Interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) are up-regulated in injured and osteoarthritic knee joints. IL-1 and TNF-α inhibit integrative meniscal repair; however, the mechanisms by which this inhibition occurs are not fully understood. Transforming growth factor-β1 (TGF-β1) increases meniscal cell proliferation and accumulation, and enhances integrative meniscal repair. An improved understanding of the mechanisms modulating meniscal cell proliferation and migration will help to improve approaches for enhancing intrinsic or tissue-engineered repair of the meniscus. The goal of this study was to examine the hypothesis that IL-1 and TNF-α suppress, while TGF-β1 enhances, cellular proliferation and migration in cell and tissue models of meniscal repair.     

Functional expression of α7 nicotinic acetylcholine receptors in human periodontal ligament fibroblasts and rat periodontal tissues

Tobacco smoking is the main risk factor associated with chronic periodontitis, but the mechanisms that underlie this relationship are largely unknown. Recent reports proposed that nicotine plays an important role in tobacco-related morbidity by acting through the nicotinic acetylcholine receptors (nAChRs) expressed by non-neuronal cells. The aim of this study was to investigate whether α7 nAChR was expressed in periodontal tissues and whether it functions by regulating IL-1β in the process of periodontitis. In vitro, human periodontal ligament (PDL) cells were cultured with 10⁻¹² M of nicotine and/or 10⁻⁹ M of alpha-bungarotoxin (α-Btx), a α7 nAChR antagonist. The expression of α7 nAChR and IL-1β in PDL cells and the effects of nicotine/α-Btx administration on their expression were explored. In vivo, an experimental periodontitis rat model was established, and the effects of nicotine/α-Btx administration on expression of α7 nAChR and development of periodontitis were evaluated. We found that α7 nAChR was present in human PDL cells and rat periodontal tissues. The expressions of α7 nAChR and IL-1β were significantly increased by nicotine administration, whereas α-Btx treatment partially suppressed these effects. This study was the first to demonstrate the functional expression of α7 nAChR in human PDL cells and rat periodontal tissues. Our results may be pertinent to a better understanding of the relationships among smoking, nicotine, and periodontitis.     

Subunit Regulation of the Human Brain α1E Calcium Channel

The α₁ subunit coding for the human brain type E calcium channel (Schneider et al., 1994) was expressed in Xenopus oocytes in the absence, and in combination with auxiliary α₂δ and β subunits. α_1E channels directed with the expression of Ba²⁺ whole-cell currents that completely inactivated after a 2-sec membrane pulse. Coexpression of α_1E with α_2bδ shifted the peak current by +10 mV but had no significant effect on whole-cell current inactivation. Coexpression of α_1E with β_2a shifted the peak current relationship by −10 mV, and strongly reduced Ba²⁺ current inactivation. This slower rate of inactivation explains that a sizable fraction (40 ± 10%, n= 8) of the Ba²⁺ current failed to inactivate completely after a 5-sec prepulse. Coinjection with both the cardiac/brain β_2a and the neuronal α_2bδ subunits increased by ≈10-fold whole-cell Ba²⁺ currents although coinjection with either β_2a or α_2bδ alone failed to significantly increase α_1E peak currents. Coexpression with β_2a and α_2bδ yielded Ba²⁺ currents with inactivation kinetics similar to the β_2a induced currents, indicating that the neuronal α_2bδ subunit has little effect on α_1E inactivation kinetics. The subunit specificity of the changes in current properties were analyzed for all four β subunit genes. The slower inactivation was unique to α_1E/β_2a currents. Coexpression with β_1a, β_1b, β₃, and β₄, yielded faster-inactivating Ba²⁺ currents than currents recorded from the α_1E subunit alone. Furthermore, α_1E/α_2bδ/β_1a; α_1E/α_2bδ/β_1b; α_1E/α_2bδ/β₃; α_1E/α_2bδ/β₄ channels elicited whole-cell currents with steady-state inactivation curves shifted in the hyperpolarized direction. The β subunit-induced changes in the properties of α_1E channel were comparable to modulation effects reported for α_1C and α_1A channels with β₃≈β_1b > β_1a≈β₄≫β_2a inducing fastest to slowest rate of whole-cell inactivation. Received: 27 March 1997/Revised: 10 July 1997     

Transforming growth factor-β1 (TGF-β1) and acetylcholine (ACh) alter nitric oxide (NO) and interleukin-1β (IL-1β) secretion in human colon adenocarcinoma cells

Colon adenocarcinoma is one of the most common fatal malignancies in Western countries. Progression of this cancer is dependent on tumor microenvironmental signaling molecules such as transforming growth factor-β (TGF-β) or acetylcholine (ACh). The present study was conducted to assess the influence of recombinant human transforming growth factor (rhTGF)-β1 or ACh on nitric oxide (NO) and interleukin-1β (IL-1β) secretion by three human colon adenocarcinoma cell lines: HT29, LS180, and SW948, derived from different grade tumors (Duke’s stage). The cells were cultured in 2D and 3D (spheroids) conditions. Colon carcinoma cells exhibited different sensitivities to rhTGF-β1 or ACh dependent on the tumor grade and the culture model. ACh exhibited significant inhibitory effects towards NO, endothelial nitric oxide synthase (eNOS), and IL-1β secretion especially by tumor cells derived form Duke’s C stage of colon carcinoma. rhTGF-β1 also decreased NO, IL-1β, and eNOS expression, but its effect was lower than that observed after the administration of ACh. The inhibition of NO and IL-1β production was more striking in 3D tumor spheroids than in 2D culture monolayers. Taken together, the TGF-β1–ACh axis may regulate colon carcinoma progression and metastasis by altering NO secretion and influence inflammatory responses by modulating IL-1β production.     

Transforming growth factor-β1 modulates the effect of 1α,25-dihydroxyvitamin D3 on leukemic cells

Summary The human leukemic cells HL-60, U937, KG-1 and THP-1 incubated with transforming growth factor-β1 (TGF-β1) were studied by examining cell surface antigens and macrophage-specific activities. The addition of 0.5 ng/ml (20 pM) of TGF-β1 with 1α,25-dihydroxyvitamin D₃ [1α, 25(OH)₂D₃] induced more Leu-M3 (CD14)-positive cells (approximately 80%) than 5×10⁻⁸ M 1α,25(OH)₂D₃ alone did (30 to 50%), although original HL-60 cells did not express any Leu-M3 antigen at all. Tumor necrosis factor-α (TNF-α) with TGF-β1 and 1α,25(OH)₂D₃ was found to potentiate the expression of these surface antigens. Furthermore, the phagocytic activity was also induced strongly. The expression of CR3 (CD11b) antigen was also increased, and all Leu-M3-positive cells were found CR3-positive when HL-60, U937, and THP-1 cells were treated with these stimulants. In contrast, CR3 but not Leu-M3 was induced in KG-1 cells after the same treatment. This may indicate that the responsiveness of leukemic cells to TGF-β1 and 1α,25(OH)₂D₃ might vary depending on a differentiation stage of the target cells. Furthermore, K562 cells originated from a more undifferentiated precursor, were not able to respond to these two inducers. These results suggested that some of TGF-β superfamily proteins might represent potent modulators in hematopoiesis, especially in the development of monocytes-macrophages or their precursors.     

Role of adhesion molecules in recruitment of Vδ1 T cells from the peripheral blood to the tumor tissue of esophageal cancer patients

 The mechanism responsible for tissue specific localization of γδ T cell subsets is not well understood. In order to explain the sequestration of specific γδ T cell subsets in the peripheral blood and tumor tissue of patients with esophageal cancer, we examined the function and expression of adhesion molecules on these cells. A hierarchy in the expression of adhesion molecules was observed. In vitro activated γδ T cells showed dominant expression of LFA-1 (CD11a), VLA-α4 (CD49d), intermediate expression of VLA-α5 (CD49e) and L-selectin (CD62L), but low expression of CD44v6 and α_Eβ₇ (CD103). It was observed that the γδ T cells use LFA-1, L-selectin and CD44v6 to bind to squamous cell carcinoma (SCC) cells, whereas they adhere to fibroblast cells using LFA-1, VLA-α4 and VLA-α5. Vδ1 T cell subsets from the peripheral blood γδ T cells utilize a larger array of adhesion molecules, namely LFA-1, VLA-α4, VLA-α5, L-selectin and α_Eβ_7, to bind to SCC cells compared to the restricted usage of LFA-1, L-selectin and CD44v6 by the Vδ2 T cells. Flow cytometric analysis of tumor infiltrating lymphocytes from the esophageal tumors confirmed the selective accumulation of Vδ1⁺γδ T cells in the tumor compartment. It thus appears that adhesion molecules expressed on these lymphocytes play an important role in the recruitment and retention of Vδ1 T cells in the tumor milieu. Received: 27 November 2000 / Accepted: 1 March 2001     

IL-1β (−511T/C) gene polymorphism not IL-1β (+3953T/C) and LT-α (+252A/G) gene variants confers susceptibility to visceral leishmaniasis

Lymphotoxin-α (LT-α) and interleukin-1beta (IL-1β) are proinflammatory cytokines playing important roles in immunity against Leishmania infection and the outcome of the disease. As cytokine productions are under the genetic control, this study tried to find any probable relationship between these cytokine gene polymorphisms and the susceptibility to visceral leishmaniasis in Iranian pediatric patients. Ninety-five pediatric patients involved with visceral leishmaniasis and 128 non-relative healthy people, from the same area as the patients, were genotyped for LT-α (+252A/G) and IL-1β (+3953T/C and −511T/C) gene polymorphisms using polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP). There was not found any significant differences in allele and genotype frequencies of LT-α (+252A/G) and IL-1β (+3953) among the study groups. However, the frequency of IL-1β −511TT genotype was higher in the controls (P = 0.0004) while the frequency of IL-1β −511CC genotype and C allele were higher in the patients (P = 0.008 and P = 0.00006, respectively). Furthermore, IL-1β CC (−511/+3953) haplotype was more frequent in VL patients compared with the controls (P = 0.0002) and the distribution of TT haplotype was higher in the controls compared with the patients (P = 0.003). In conclusion, based on the results, IL-1β −511C allele, CC genotype and CC (−511/+3953) haplotype could be considered as the susceptibility factors for visceral leishmaniasis while IL-1β −511TT genotype, T allele and TT haplotype (−511/+3953) might be counted as the influential factors for resistance to the disease.     

In vitro cytokine stimulation assay for glycolipid biosurfactant from <Emphasis Type="Italic">Rhodococcus</Emphasis><Emphasis Type="Italic">ruber</Emphasis>: role of monocyte adhesion

Glycolipid biosurfactant (GLB) from Rhodococcus ruber IEGM 231 was found to stimulate tumor necrosis factor-α (TNF-α), interleukin (IL) -1β and IL-6 production when applied as an ultrasonic emulsion to the adherent human peripheral blood monocyte culture. However, a lack of cytokine-stimulating activity was registered with the GLB applied as a hydrophobic film coating in 24-well culture plates, indicating that it may have been due to its inhibitory effect on monocyte adhesion. The mode of GLB application may therefore play an important role in in vitro assay of immunostimulatory activity of this compound as well as other bacterial glycolipids. Additionally, GLB from R. ruber displayed no cytotoxicity against human lymphocytes and therefore could be proposed as a potential immunomodulating and antitumor agent.     

Levamisole regulates the proliferation of murine liver T cells through Kupffer-cell-derived cytokines

 We have previously shown that levamisole increases the cytotoxic, cytostatic, and proliferative activity of murine nonparenchymal liver cells (NPC) in vitro. We have also shown that the nonadherent subpopulation of NPC, which are composed predominantly of T lymphocytes, is very responsive to this agent when administered to mice. Kupffer cells or immigrant macrophages are also responsive to levamisole but to a lesser extent. These findings prompted us to investigate changes in cytokine production by NPC following-treatment of mice with levamisole (25 mg/kg, i.p.), which may help explain the observed alterations in the immune functions of these cells. We found that levamisole treatment of mice causes a threefold increase in production of interferon (IFN) α/β by adherent NPC (more than 80% – 90% Kupffer cells) in vitro. When IFN α/β was added to cultured cells, it decreased the proliferative capacity of liver T cells in a dose-dependent manner. In contrast, the addition of anti-IFNα/β was shown to augment levamisole-induced proliferation of unfractionated NPC and Kupffer cells. NPC production of interleukin 1 (IL-1) and interleukin-6 (IL-6) in vitro was also increased threefold following treatment of mice with levamisole. IL-6 added in vitro to cells significantly augmented levamisole-induced proliferation of liver T cells while anti-IL-6 reduced proliferative activity to control levels. These findings suggested that IFNα/β, IL-6, and IL-1 play important regulatory roles in controlling the proliferative response of murine liver-associated T lymphocytes to levamisole. Finally, the proliferation of bone marrow cells was increased in mice given 5-fluorouracil (5FU). On the other hand, the proliferation of NPC was dramatically suppressed when 5FU was administered. However, the proliferation of these cells was restored when levamisole was given after 5FU. Received: 27 November 1995 / Accepted: 16 October 1996     

Regulation of melanin synthesis by the TGF-β family in B16 melanoma cells

Effects of representative members of the transforming growth factor-β (TGF-β) family, TGF-β1, activin A and BMP-2, on melanin content and expression of pigment-producing enzymes were examined in B16 melanoma cells. Treatment with TGF-β1 or activin A but not with BMP-2 significantly decreased melanin content and expression of Tyrosinase and Tyrp-1, suggesting an inhibitory effect of TGF-β1 and activin A on melanin synthesis. TGF-β1 completely inhibited melanin synthesis induced by α-melanin stimulating hormone (α-MSH), whereas activin A only slightly did. As compared with parental B16 cells, the inhibitory effects of TGF-β1 and activin A on melanin content were relative smaller in B16 F10 cells, a subline of B16 cells that contain more pigment. The present study indicates that in addition to TGF-β, activin negatively regulates melanogenesis in the absence of α-MSH, but that the activity in the presence of α-MSH was slightly different between TGF-β and activin.