Inflammatory cytokines IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha impart neuroprotection to an excitotoxin through distinct pathways.

The proinflammatory cytokines IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha are produced within the CNS, and, similar to the periphery, they have pleotrophic and overlapping functions. We have shown previously that TNF-alpha increases neuronal survival to a toxic influx of calcium mediated through neuronal N-methyl-d -aspartic acid (NMDA) glutamate-gated ion channels. This process, termed excitotoxicity, is a major contributor to neuronal death following ischemia or stroke. Neuroprotection by this cytokine requires both activation of the p55/TNF receptor type I and the release of TNF-alpha from neurons, and it is inhibited by the plant alkaloid nicotine. Here, we report that other inflammatory cytokines (IL-1 alpha, IL-1 beta, and IL-6) are also neuroprotective to excessive NMDA challenge in our system. Neuroprotection provided by IL-1 is distinct from TNF-alpha because it is inhibited by IL-1 receptor antagonist; it is not antagonized by nicotine, but it is inhibited by a neutralizing Ab to nerve growth factor (NGF). Similar to IL-1, IL-6-mediated neuroprotection is also antagonized by pretreatment with IL-1 receptor antagonist and it is not affected by nicotine. However, neutralizing anti-NGF only partially blocks IL-6-mediated protection. These studies support an important role for distinct but overlapping neuroprotective cytokine effects in the CNS.     

Blood concentrations of the cytokines IL-1beta,IL-6, IL-10, TNF-alpha and IFN-gamma during experimentally induced swine dysentery

Background  

Knowledge of the cytokine response at infection with Brachyspira hyodysenteriae can help understanding disease mechanisme involved during swine dysentery. Since this knowledge is still limited the aim of the present study was to induce dysentery experimentally in pigs and to monitor the development of important immunoregulatory cytokines in blood collected at various stages of the disease.     

Effects of IL-10 and age on IL-6, IL-1beta, and TNF-alpha responses in mouse skeletal and cardiac muscle to an acute inflammatory insult.

Exaggerated proinflammatory cytokine responses can be observed with aging, and reduced levels of the anti-inflammatory cytokine IL-10 may contribute to these responses. IL-10 can reduce IL-6, IL-1beta, and TNF-alpha expression in nonmuscle tissues; however, no studies have examined the combined effects of IL-10 and age on cytokine responses in skeletal and cardiac muscle. These experiments tested the hypothesis that the absence of IL-10, in vivo, is associated with greater IL-6, TNF-alpha, and IL-1beta responses to an inflammatory challenge in skeletal and cardiac muscle and that aging exaggerates these responses. We compared IL-6, IL-1beta, and TNF-alpha mRNA and protein levels in skeletal and cardiac muscle of young (4 mo) and mature (10-11 mo) wild-type (IL-10(+/+)) and IL-10 deficient (IL-10(-/-)) mice following LPS. Skeletal and cardiac IL-6 mRNA and protein were elevated by LPS for IL-10(+/+) and IL-10(-/-) mice with greater responses in the IL-10(-/-) mice (P < 0.01). In skeletal muscle these effects were greater in mature than young mice (P < 0.01). IL-1beta mRNA and protein responses to LPS were greater in cardiac muscle of young but not mature IL-10(-/-) mice compared with IL-10(+/+) (P < 0.01). However, IL-1beta responses were greater in mature than young mice, but only in IL-10(+/+) groups (P < 0.05). The absence of IL-10 was associated with higher TNF-alpha protein levels in cardiac muscle (P < 0.05). The results provide the first in vivo evidence that the absence of IL-10 is associated with a greater IL-6 response to LPS in skeletal and cardiac muscles, and in skeletal muscle aging further exaggerates these responses.     

Changes in plasma IL-1beta, TNF-alpha and IL-6 after total hip replacement surgery in general or regional anaesthesia

Different anaesthetic methods influence the neuro-immuno-endocrine biologic responses to surgery and may thus possibly interfere with the postoperative course and development of complications. The neuroendocrine system is closely related to the cytokine network. In this study, the effects of general anaesthesia (n=6) and regional spinal/epidural anaesthesia (n=6) on the cytokine response (IL-1beta, TNFalpha, IL-6) to uncemented total hip replacement surgery were evaluated. The postoperative clinical course was uneventful in every case. In both groups, only very low values of plasma IL-beta were measured perioperatively, whereas plasma IL-6 increased postoperatively with peak values 4 h after surgery. The changes in plasma TNF-alpha were not significant. No significant differences in plasma TNF-alpha or IL-6 were found between patients operated in general or in regional anaesthesia. This suggests minor influence of plasma cytokines on the possible beneficial effects of regional anaesthesia on the clinical course after surgery in low risk patients. There were slightly higher TNF-alpha and IL-6 levels after the operation and significantly lower cortisol levels during the operation in the regional anaesthesia group compared to the general anaesthesia group, giving rise to a significant inverse correlation between peak values of IL-6 and peak values of cortisol. This supports the theory that after surgery the inhibitory effect of cortisol on monocyte cytokine production overrides adrenergic stimulation.     

Recombinant human IL-1 beta and TNF-alpha stimulate production of IL-1 alpha and IL-1 beta by vascular smooth muscle cells and IL-1 alpha by vascular endothelial cells

Vascular endothelial cells (EC) produced IL-1 alpha but not IL-1 beta into extracellular fluids. Vascular smooth muscle cells (SMC), on the other hand, produced both IL-1 alpha and IL-1 beta, and IL-1 beta produced was much higher than IL-1 alpha. The addition of recombinant human IL-1 beta or recombinant human TNF-alpha significantly enhanced IL-1 alpha production in EC, and IL-1 alpha and IL-1 beta production in SMC. IL-1 beta release was not observed even when EC were stimulated with TNF-alpha. These results suggest that the species of released form of IL-1 are different in different cell types and that cytokines enhance IL-1 alpha and IL-1 beta production in SMC and IL-1 alpha production in EC.     

The importance of IL-1 beta and TNF-alpha,and the noninvolvement of IL-6, in the development of monoclonal antibody-induced arthritis

Injection of anti-type II collagen Ab and LPS induces arthritis in mice. The levels of IL-1 beta, IL-6, and chemokines (macrophage inflammatory protein (MIP)-1 alpha, MIP-2, and monocyte chemoattractant protein-1) in the hind paws increased with the onset of arthritis and correlated highly with arthritis scores. The level of TNF-alpha was also elevated, but only transiently. Quantitative real-time PCR analysis revealed increases in cytokine and chemokine mRNA. To elucidate the contribution of inflammatory cytokines and chemokines in arthritis development more directly, recombinant proteins, neutralizing Abs, and knockout mice were used. The injection of rIL-1 beta or TNF-alpha, but not IL-6 or chemokines, induced arthritis when mice were i.v. preinjected with anti-type II collagen Ab. However, a single injection of recombinant cytokines or chemokines into the hind paws did not induce swelling. Arthritis development was inhibited by neutralizing Ab against IL-1 beta, TNF-alpha, or MIP-1 alpha. In contrast, the inhibitory effect by anti-MIP-2 Ab was partial and, surprisingly, Abs to IL-6 and monocyte chemoattractant protein-1 showed no inhibitory effect. Furthermore, arthritis development in IL-1R(-/-) mice and TNFR(-/-) mice was not observed at all, but severe arthritis was developed in IL-6(-/-) mice. These results suggest that IL-1 beta and TNF-alpha play more crucial roles than IL-6 or chemokines in this model. Because arthritis was also developed in SCID mice, the development of arthritis in the Ab-induced mice model is due to a mechanism that does not involve T or B cells.     

Identification of rat IL-1beta, IL-2, IFN-gamma and TNF-alpha in activated splenocytes by intracellular immunostaining.

We have developed a sensitive three-step indirect immunofluorescence method to identify individual rat cells that produce cytokines including IL-1beta, IL-2, IFN-gamma and TNF-alpha. Cultured rat splenocytes were polyclonally activated to cytokine synthesis by mitogens such as lipopolysaccharide or a combination of a protein kinase C activator (phorbol 12-myristate 13-acetate) and a calcium ionophore (ionomycin). Careful selection of either antigen affinity-purified polyclonal or monoclonal cytokine-detecting antibodies combined with gentle fixation and permeabilization of the cells enabled discrimination of cytokine-producing cells based on distinct morphological staining criteria. Cells making IL-2, IFN-gamma and TNF-alpha could be identified by a characteristic, intracellular, rounded, juxtanuclear immunofluorescence signal. This staining pattern reflected the accumulation of the intracellularly processed cytokines in the Golgi organelle of producer cells. The staining of cells that synthesized IL-1beta, which is not transported intracellularly via the endoplasmatic reticulum-Golgi pathway, generated a different, but distinct and reproducible staining pattern, IL-1beta producing macrophages expressed intense nuclear immunofluorescence with additional reticular, cytoplasmic signals. Furthermore, the use of biologically neutralizing detecting antibodies against the cytokines under study prevented recognition of surface-stained target cells that had bound secreted cytokines by cytokine-specific receptors. This modified staining technology enabled analysis of the kinetic pattern and the frequency of cytokine-producing cells in cultures of rat splenocytes after various modes of polyclonal activation.     

Serum IL-1beta, IL-2, and IL-6 in insulin-dependent diabetic children

Insulin-dependent diabetes mellitus (IDDM) is a chronic disease characterized by T-cell-dependent autoimmune destruction of the insulin-producing beta cells in the pancreatic islets of Langerhans, resulting in an absolute lack of insulin. T cells are activated in response to islet-dominant autoantigens, the result being the development of IDDM. Insulin is one of the islet autoantigens responsible for the activation of T-lymphocyte functions, inflammatory cytokine production, and development of IDDM. The aim of this study was to investigate serum concentrations of interleukin (IL)-1beta, IL-2, IL-6, and tumor necrosis factor (TNF)-alpha in children IDDM. The study population consisted of 27 children with IDDM and 25 healthy controls. Children with IDDM were divided into three subgroups: (1) previously diagnosed patients (long standing IDDM) (n : 15), (2) newly diagnosed patients with diabetic ketoacidosis (before treatment) (n : 12), and (3) newly diagnosed patients with diabetic ketoacidosis (after treatment for two weeks) (n : 12). In all stages of diabetes higher levels of IL-1beta and TNF-alpha and lower levels of IL-2 and IL-6 were detected. Our data about elevated serum IL-1beta, TNF-alpha and decreased IL-2, IL-6 levels in newly diagnosed IDDM patients in comparison with longer standing cases supports an activation of systemic inflammatory process during early phases of IDDM which may be indicative of an ongoing beta-cell destruction. Persistence of significant difference between the cases with IDDM monitored for a long time and controls in terms of IL-1beta, IL-2, IL-6, and TNF-alpha supports continuous activation during the late stages of diabetes.     

IL-6, RANKL, TNF-alpha/IL-1: interrelations in bone resorption pathophysiology

All osteogenic cells (osteoclasts, osteoblasts) contribute individually to bone remodeling. Their cellular interactions control their cellular activities and the bone remodeling intensity. These interactions can be established either through a cell-cell contact, involving molecules of the integrin family, or by the release of many polypeptidic factors and/or their soluble receptor chains. These factors can act directly on osteogenic cells and their precursors to control differentiation, formation and functions (matrix formation, mineralization, resorption...). Here, we present the involvement of three groups of cytokines which seem to be of particular importance in bone physiology: interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) (TNF-alpha)/IL-1, and the more recently known triad osteoprotegerin (OPG)/receptor activator of NF-kappaB (RANK)/RANK ligand (RANKL). The interactions between these three groups are presented within the framework of bone resorption pathophysiology such as tumor associated osteolysis. The central role of the OPG/RANK/RANKL triad is pointed out.     

Involvement of PKC-beta in PTH, TNF-alpha, and IL-1 beta effects on IL-6 promoter in osteoblastic cells and on PTH-stimulated bone resorption

Protein kinase C (PKC) has been shown to be activated by parathyroid hormone (PTH) in osteoblasts. Prior evidence suggests that this activation mediates responses leading to bone resorption, including production of the osteoclastogenic cytokine interleukin-6 (IL-6). However, the importance of specific PKC isozymes in this process has not been investigated. A selective antagonist of PKC-beta, LY379196, was used to determine the role of the PKC-beta isozyme in the expression of IL-6 in UMR-106 rat osteoblastic cells and in bone resorption in fetal rat limb bone organ cultures. PTH, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta) induced translocation of PKC-alpha and -beta(I) to the plasma membrane in UMR-106 cells within 5 min. The stimulation of PKC-beta(I) translocation by PTH, TNF-alpha or IL-1 beta was inhibited by LY379196. In contrast, LY379196 did not affect PTH, TNF-alpha-, or IL-1 beta-stimulated translocation of PKC-alpha. PTH, TNF-alpha, and IL-1 beta increased luciferase expression in UMR-106 cells transiently transfected with a -224/+11 bp IL-6 promoter-driven reporter construct. The IL-6 responses were also attenuated by treatment with LY379196. Furthermore, LY379196 inhibited bone resorption elicited by PTH in fetal rat bone organ cultures. These results indicate that PKC-beta(I) is a component of the signaling pathway that mediates PTH-, TNF-alpha-, and IL-1 beta-stimulated IL-6 expression and PTH-stimulated bone resorption.     

Age-related alterations in IL-1beta, TNF-alpha, and IL-6 concentrations in parotid acinar cells from BALB/c and non-obese diabetic mice.

IL-1beta, TNF-alpha, and IL-6 have been implicated in the destruction of parotid gland acinar cells (but not duct cells) in autoimmune sialoadenitis. Here we report the temporal alterations of these cytokines in parotid acinar cells that may lead to this specificity in cell death in the non-obese diabetic (NOD) mouse model for Sj?gren's syndrome. Immunohistochemistry on paraffin sections of parotid gland from 5- and 10-week-old BALB/c and NOD mice confirmed the presence of many peri-acinar lymphoid nodules but few T-cells and macrophages between acinar cells. RT-PCR on enzymatically dispersed mouse parotid acinar cells (MPACs) showed no bands for CD3varepsilon, CD20, or F4/80 regardless of mouse strain or age. By ELISA, MPACs from 10-week-old NODs showed a small but highly significant (p<0.003) increase in IL-1beta and a large significant decrease (p<0.008) in IL-6 compared to 5-week-old NODs. Norepinephrine-stimulated amylase release from MPACs was not different regardless of mouse strain or age. These data show that alterations in acinar cell production of IL-1beta and IL-6 in aging NODs precede periductal lymphoid aggregates and acinar cell secretory dysfunction. (J Histochem Cytochem 48:1033-1041,2000)     

Pravastatin immunomodulates IL-6 and C-reactive protein, but not IL-1 and TNF-alpha, in cardio-pulmonary bypass

BACKGROUND: While statins are increasingly used in cardiopulmonary bypass (CPB), the anti-inflammatory effects of individual statins, within the context of various treatment regimes, need further examination. The present study evaluates the anti-inflammatory effectiveness of the short-term, preoperative and intensive postoperative use of pravastatin in CPB. METHOD: Forty three patients undergoing CPB were enrolled in a randomized, prospective clinical study. One group (n = 21), received pravastatin, the other (n = 22) did not. Patients in the pravastatin group received one dose of 40 mg per day for nine days, starting 48 hours before CPB, with an additional dose of 40 mg one hour after surgery. Plasma levels of selected inflammatory mediators were measured at baseline and tracked systematically. RESULTS: Pravastatin reduced postoperative interleukin-6 (IL-6) levels significantly at 24 and 48 hours, and at seven days. Mean +/- SD values, for treated versus untreated patients were: at 24 hours, 159.5 +/- 58.5 versus 251.2 +/- 53.0 pg/mL (p < 0.001); at 48 hours, 81.9 +/- 31.5 versus 194.2 +/- 56.3 pg/mL (p < 0.001); and at seven days, 16.4 +/- 7.2 versus 30.8 +/- 12.6 (p < 0.001). C-reactive protein (CRP) decreased significantly on the seventh postoperative day, when plasma levels were 3.6 +/- 1.1 in the treated patients versus 8.2 +/- 2.1 mg/dL in the controls (p < 0.001). No changes in plasma IL-1 and TNF-alpha were found during entire study. CONCLUSIONS: Pravastatin induced a precocious modulation of IL-6 expression and a later reduction of plasma CRP levels. Pravastatin;s effects on the expression of these pivotal inflammatory mediators strongly support its well-timed use in CPB.     

Plasminogen-induced IL-1beta and TNF-alpha production in microglia is regulated by reactive oxygen species

Microglia, major immune effector cells in the central nervous system, become activated during brain injury. In this study we showed that the blood component plasminogen/plasmin activates microglia. Plasminogen-induced IL-1beta, TNF-alpha, and iNOS mRNA expression in primary cultured rat microglia and BV2 murine microglial cells. Plasmin caused a similar response. Serine protease inhibitors suppressed both plasminogen- and plasmin-induced IL-1beta and TNF-alpha expression, indicating the importance of serine protease activity in plasminogen/plasmin activation of microglia. Reactive oxygen species (ROS) appeared to play an important role in plasminogen-induced microglial activation, with ROS being generated within 15min of plasminogen treatment, and antioxidants (100 microM trolox and 10mM NAC) reducing IL-1beta and TNF-alpha expression in plasminogen-treated cells. Furthermore, plasminogen stimulated CREB and NF-kappaB DNA binding activity, and this activation was also reduced by trolox and NAC. These results suggest that plasminogen activates microglia via stimulation of ROS production.     

Neutrophil gelatinase-associated lipocalin is up-regulated in human epithelial cells by IL-1 beta, but not by TNF-alpha

Synthesis of the antimicrobial protein neutrophil gelatinase-associated lipocalin (NGAL) increases dramatically in bronchial epithelial cells and alveolear type II pneumocytes during lung inflammation. IL-1beta induces a >10-fold up-regulation of NGAL expression in the type II pneumocyte-derived cell line A549 cells, whereas TNF-alpha, IL-6, and LPS had no effect. Similar IL-1beta selectivity was demonstrated in primary bronchial epithelial cells and epidermal keratinocytes and for an NGAL promoter fragment transfected into A549 cells. By deletion and substitution analysis of the NGAL promoter, a 40-bp region containing an NF-kappaB consensus site was found to control the IL-1beta-specific up-regulation. Involvement of the NF-kappaB site was demonstrated by site-directed mutagenesis, by transfection with a dominant-negative inhibitor of the NF-kappaB pathway, and by EMSA. TNF-alpha activation of NF-kappaB, in contrast, did not increase NGAL synthesis, even though induced binding of NF-kappaB to the NGAL promoter was observed in vitro. IL-1beta specificity was not contained within the NF-kappaB site of the NGAL promoter, as determined by exchanging the NGAL promoter's NF-kappaB-binding sequence with that of the IL-8 promoter or with the NF-kappaB consensus sequence and by testing the NF-kappaB-binding sequence of the NGAL promoter against the heterologous SV40 promoter. Selectivity for the IL-1 pathway was substantiated by demonstrating that NGAL promoter activity could be induced by LPS stimulation of A549 cells transiently expressing Toll-like receptor 4, which use the same intracellular signaling pathway as the IL-1R. Together, this demonstrates a selective up-regulation of NGAL by the IL-1 pathway.     

Direct influences of pro-inflammatory cytokines (IL-1beta, TNF-alpha, IL-6) on the proliferation and cell-surface antigen expression of cancer cells

Pro-inflammatory cytokines, e.g. interleukin 1 (IL-1), tumour necrosis factor alpha (TNF-alpha), IL-6 produced by surgical intervention or non-specific immunotherapy may directly affect both the growth and the metastasis of tumour cells. It is therefore important to clarify the direct influence of pro-inflammatory cytokines on tumour cells in order to obtain a better knowledge of anti-tumour therapy. Four human lung cancer cell lines were used. The tumour cells were incubated for 72 h in the presence of various concentrations of IL-1beta, TNF-alpha, or IL-6 and then the proliferative response was assessed by an MTT assay. After 14 days of culture with each pro-inflammatory cytokine, the cell-surface antigen expressions (HLA-class I, HLA-class II, CEA, sialyl Lewis(x)) were assessed by an immunocytochemical staining method. Among the various combinations of tumour cells (PC-9, PC-12, QG-56, QG-95) and cytokines (IL-1beta, TNF-alpha, IL-6), only TNF-alpha significantly exhibited an antiproliferative effect against PC-9 cells. However, various modulations of the cell-surface antigen expression by the cytokines were observed. The HLA-class I antigen expression of PC-9 was augmented by either TNF-alpha or IL-1beta. Furthermore, IL-1beta was able to induce CEA in PC-9, QG-56, and QG-95 cells while TNF-alpha was able to enhance the expression of sialyl Lewis(x)in QG-95 cells. Although the influence of pro-inflammatory cytokines on the growth of tumour cells was only slight, some modulations of the cell-surface antigen expression were notable. The augmentation of HLA-class I expression can thus improve the immunogenicity of tumour cells while the induction of CEA or sialyl Lewis(x)may therefore be associated with the promotion of metastasis.     

IL-1beta and TNF-alpha induce increased expression of CCL28 by airway epithelial cells via an NFkappaB-dependent pathway

CCL28 is a mucosal chemokine that attracts eosinophils and T cells via the receptors CCR3 and CCR10. Consequently, it is a candidate mediator of the pathology associated with asthma. This study examined constitutive and induced expression of CCL28 by A549 human airway epithelial-like cells. Real-time RT-PCR and ELISA of cultured cells and supernatants revealed constitutive levels of CCL28 expression to be low, whereas IL-1beta and TNF-alpha, induced significantly increased expression. Observations from induced sputum and human airway biopsies supported this. Signal transduction studies revealed that IL-1beta and TNF-alpha stimulation induced NFkappaB phosphorylation in A549 cells, but antagonist inhibition of NFkappaB p50-p65 phosphorylation correlated with marked reduction of IL-1beta or TNF-alpha induced CCL28 expression. Together these studies imply a role for CCL28 in the orchestration of airway inflammation, and suggest that CCL28 is one link between microbial insult and the exacerbation of pathologies such as asthma, through an NFkappaB-dependent mechanism.     

Opiate regulation of IL-1beta and TNF-alpha in cultured human articular chondrocytes

In order to investigate if beta-endorphins anti-inflammatory effect in cartilage-damaging states is mediated via tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), we examined its influence on these two cytokines in vitro. Human articular chondrocytes were obtained from patients undergoing total knee arthroplasty and stimulated with beta-endorphin (60-6000 ng/ml). Protein levels of TNF-alpha and IL-1 beta were measured by ELISA in supernatants from articular chondrocyte cultures. beta-Endorphin significantly increased the levels of IL-1 beta for all concentrations used after 15 min incubation, and when stimulated with 600 and 6000 ng/ml after 24 h incubation. The opioid-induced increase in IL-1 beta was blocked by naltrexone in the group tested. TNF-alpha expression was also significantly stimulated by 60 and 600 ng/ml beta-endorphin after 15 min, an effect blocked by naltrexone in the group tested. These findings indicate that the mechanism of beta-endorphins anti-inflammatory influence in cartilage-damaging states is not apparently mediated via these two cytokines modulation.