Interleukin-1beta mRNA Levels Research Articles

Melatonin Alleviates Acute Gouty Inflammation In Vivo and In Vitro.

The aim of this study was to identify the effects of melatonin on acute gouty inflammation and to investigate the underlying mechanisms. We found significantly lower serum melatonin levels in gout patients in the acute phase than in those in the remission phase or in normal individuals. The mRNA expression of melatonin receptor 2 (MT2) was also lower in gout patients than in normal individuals. To verify the in-vivo role of melatonin, a gouty arthritis model was established by intraarticular injection of monosodium urate (MSU, 1 mg) crystals into the paws of C57BL/6 mice. Joint inflammation in the mouse model was evaluated by measuring the thickness of the right paw/left paw, and the inflammation index was determined by examining infiltrating neutrophils with haematoxylin and eosin (H&E) staining. Melatonin was found to reduce both paw thickness and the inflammation index in the mouse model, and melatonin also reduced the mRNA levels of interleukin-1 beta (IL-1β), IL-6 and NLR family pyrin domain containing 3 (NLRP3) inflammasome. To mimic gouty inflammation in vitro, mouse peritoneal macrophages were stimulated with lipopolysaccharides (LPS) plus MSU. Melatonin was revealed to reduce IL-1β secretion by stimulated macrophages. The mRNA expression levels of IL-1β and IL-6 were also inhibited by melatonin. Western blot analysis showed that the expression of NLRP3, caspase-1 and pro-IL-1β was also inhibited by melatonin. In conclusion, our study demonstrated that melatonin alleviated gouty inflammation in vivo and in vitro, and the underlying mechanism may involve inhibiting the assembly of the NLRP3 inflammasome.

IL1B Increases Intestinal Tight Junction Permeability by Up-regulation of MIR200C-3p, Which Degrades Occludin mRNA

Defects in the epithelial tight junction (TJ) barrier contribute to development of intestinal inflammation associated with diseases. Interleukin 1 beta (IL1B) increases intestinal permeability in mice. We investigated microRNAs that are regulated by IL1B and their effects on expression of TJ proteins and intestinal permeability. We used Targetscan to identify microRNAs that would bind the 3' untranslated region (3'UTR) of occludin mRNA; regions that interacted with microRNAs were predicted using the V-fold server and Assemble2, and 3-dimensional models were created using UCSF Chimera linked with Assemble2. Caco-2 cells were transfected with vectors that express microRNAs, analyzed by immunoblots and real-time polymerase chain reaction (PCR), and grown as monolayers; permeability in response to IL1B was assessed with the marker inulin. Male C57BL/6 mice were given intraperitoneal injections of IL1B and intestinal recycling perfusion was measured; some mice were given dextran sodium sulfate to induce colitis and/or gavage with an antagonist to MIR200C-3p (antagomiR-200C) or the nonspecific antagomiR (control). Intestinal tissues were collected from mice and analyzed by histology and real-time PCR; enterocytes were isolated by laser capture microdissection. We also analyzed colon tissues and organoids from patients with and without ulcerative colitis. Incubation of Caco-2 monolayers with IL1B increased TJ permeability and reduced levels of occludin protein and mRNA without affecting the expression of other transmembrane TJ proteins. Targetscan identified MIR122, MIR200B-3p, and MIR200C-3p, as miRNAs that might bind to the occludin 3'UTR. MIR200C-3p was rapidly increased in Caco-2 cells incubated with IL1B; the antagomiR-200c prevented the IL1B-induced decrease in occludin mRNA and protein and reduced TJ permeability. Administration of IL1B to mice increased small intestinal TJ permeability, compared with mice given vehicle; enterocytes isolated from mice given IL1B had increased expression of MIR200C-3p and decreased levels of occludin messenger RNA (mRNA) and protein. Intestinal tissues from mice with colitis had increased levels of IL1B mRNA and MIR200C-3p and decreased levels of occludin mRNA; gavage of mice with antagomiR-200C reduced levels of MIR200C-3p and prevented the decrease in occludin mRNA and the increase in colonic permeability. Colon tissues and organoids from patients with ulcerative colitis had increased levels of IL1B mRNA and MIR200C-3p compared with healthy controls. Using 3-dimensional molecular modeling and mutational analyses, we identified the nucleotide bases in the occluding mRNA 3'UTR that interact with MIR200C-3p. Intestine tissues from patients with ulcerative colitis and mice with colitis have increased levels of IL1B mRNA and MIR200C-3p, which reduces expression of occludin by enterocytes and thereby increases TJ permeability. Three-dimensional modeling of the interaction between MIR200C-3p and the occludin mRNA 3'UTR identified sites of interaction. The antagomiR-200C prevents the decrease in occludin in enterocytes and intestine tissues of mice with colitis, maintaining the TJ barrier.

In vitro immunotoxicological effects of heavy metals on European sea bass (Dicentrarchus labrax L.) head-kidney leucocytes.

The knowledge about the direct effects of heavy metals on fish leucocytes is still limited. We investigate the in vitro effects of heavy metals (Cd, Hg, Pb or As) on oxidative stress, viability and innate immune parameters of head-kidney leucocytes (HKLs) from European sea bass (Dicentrarchus labrax). Production of free oxygen radicals was induced by Cd, Hg and As, mainly after 30 min of exposure. Cd and Hg promoted both apoptosis and necrosis cell death while Pb and As did only apoptosis, in all cases in a concentration-dependent manner. Moreover, expression of genes related to oxidative stress and apoptosis was significantly induced by Hg and Pb but down-regulated by As. In addition, the expression of the metallothionein A gene was up-regulated by Cd and Pb exposure though this transcript, as well as the heat shock protein 70, was down-regulated by Hg. Cd, methylmercury (MeHg) and As reduced the phagocytic ability, whereas Hg and Pb increased it. Interestingly, all the heavy metals decreased the phagocytic capacity (the number of ingested particles per cell). Leucocyte respiratory burst changed depending on the metal exposure, usually in a time- and dose-manner. Interestingly, the expression of immune-related genes was slightly affected by Cd, MeHg, As or Pb being Hg the form producing the greatest alterations, which included down-regulation of immunoglobulin M and hepcidin, as well as the up-regulation of interleukin-1 beta mRNA levels. This study provides an in vitro approach for elucidating the heavy metals toxicity, and particularly the immunotoxicity, in fish leucocytes.

Effect of sodium butyrate on intestinal inflammatory response to lipopolysaccharide in broiler chickens

Jiang, Y., Zhang, W., Gao, F. and Zhou, G. 2015. Effect of sodium butyrate on intestinal inflammatory response to lipopolysaccharide in broiler chickens. Can. J. Anim. Sci. 95: 389–395. The aim of this study was to investigate the effect of sodium butyrate (SB) supplementation on intestinal inflammatory response to lipopolysaccharide (LPS) in broiler chickens. A total of 120 one-day-old chickens (Arbor Acres) were divided into two groups and fed a control diet (without SB) or 1.00 g SB kg−1diet. Half of the chickens fed each diet were injected intra-peritoneally with 0.5 g kg−1body weight of Escherichia coli LPS at 16, 18 and 20 d of age. The results showed that the LPS challenge decreased (P<0.05) villus height and the ratio of villus height to crypt depth (V/C ratio), increased (P<0.01) crypt depth of the duodenum. SB supplementation increased (P<0.05) villus height, crypt depth and V/C ratio of the duodenum and jejunum, except for the crypt depth of the jejunum. The LPS challenge increased (P<0.05) myeloperoxidase (MPO) activities, intercellular adhesion molecule-1 (ICAM-1) and activated nuclear factor kappa B (NF-κB) levels in mucosa of the duodenum and jejunum, while decreasing (P<0.05) insulin-like growth factor-1 (IGF-1) concentrations. The LPS challenge increased (P<0.05) the mRNA levels of interleukin-1beta (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in duodenal mucosa. SB supplementation decreased (P<0.05) the MPO activities and mRNA expression of TNF-α in the duodenal mucosa, and the activated NF-κB levels in mucosa of the duodenum and jejunum. There were no significant interactions between dietary SB and LPS on the histomorphology of the small intestine and those inflammatory mediators except for MPO and ICAM-1 in duodenal mucosa (P<0.05). The results indicate that SB supplementation could improve the intestinal morphology and function of broiler chickens and partially attenuate inflammatory responses caused by LPS challenge but not involving NF-κB activation.

Early-stage blocking of Notch signaling inhibits the depletion of goblet cells in dextran sodium sulfate-induced colitis in mice

Goblet cells, which contribute to mucosal defense and repair in the intestinal epithelium, are depleted in human and rodent colitis. The Notch signal pathway regulates the differentiation of intestinal stem cells into epithelial cells and inhibits the differentiation of secretory lineages, including goblet cells. The aim of our study was to clarify whether the blocking of the Notch pathway at an early stage of colitis would preserve goblet cells and facilitate the healing process in dextran sulfate sodium (DSS)-induced colitis in mice. DSS was orally administered to C57/BL6 mice for 7 days, and dibenzazepine (DBZ), a Notch pathway blocker, was administered for 5 consecutive days, beginning on the first day of DSS treatment. Colonic mucosal inflammation was evaluated clinically, biochemically, and histologically. The expression of the goblet cell-associated genes Math1 and MUC2 and proinflammatory cytokines was evaluated by real-time reverse-transcriptase-PCR, with the expression of Math1 and MUC2 also visualized by immunohistochemical examination. The administration of DBZ at 4 mumol/kg significantly reduced the severity of the colitis. Compared with the DSS only-treated intestine, the number of goblet cells was relatively sustained, and the expression of Math1 and MUC2 was also elevated in the DSS/DBZ-treated intestine. DBZ treatment suppressed the mRNA levels for interleukin-1beta and -6, and matrix metalloproteinases-3 and -9 in the DSS-treated intestine. Early-stage blocking of Notch signaling may ameliorate acute DSS colitis by preventing reduction in the number of goblet cells.

Influence of age on behavioral, immune and endocrine responses to the T‐cell superantigen staphylococcal enterotoxin A

Aged subjects are more vulnerable to administration of the endotoxin lipopolysaccharide, but research on age-associated sensitivity to other immune stimulants has been limited. The current study examined the effects of administering the superantigen, staphylococcal enterotoxin A (SEA), to young (4-month-old) and aged (20-month-old) male C57BL/6J mice on consumption of a novel liquid, cytokine production, corticosterone levels, and expression of central mRNA levels of cytokines and corticotropin-releasing hormone. SEA produced exaggerated hypophagia in aged mice, as they showed decreased consumption that persisted for 24 h. SEA increased hypothalamic mRNA levels of interleukin-1beta in the aged, but not the young, mice 2 h after administration. No differences in cytokine expression were observed 24 h after SEA. Both age groups showed increased plasma corticosterone levels 2 h after SEA administration. However, 24 h after SEA exposure the aged, but not the young, mice showed an augmented corticosterone response to the consumption test. Collectively, these data show that aging may exacerbate the behavioral and neuroinflammatory response to superantigen exposure. Further, the present study suggests that immune activation may result in delayed alterations in stress-induced corticosterone production in aged subjects.

Leptin regulates leukocyte recruitment into the brain following systemic LPS-induced inflammation

The appetite suppressing hormone leptin has emerged as an important modulator of immune function and is now considered to be a critical link between energy balance and host defense responses to pathogens. These 'adaptive' responses can, in situations of severe and sustained systemic inflammation, lead to adverse effects including brain damage that is partly mediated by neutrophil recruitment into the brain. We examined the contribution of leptin to this process in leptin-deficient (ob/ob), -resistant (db/db) and wild-type (WT) mice injected intraperitoneally with a septic dose of lipopolysaccharide (LPS). This treatment induced a dramatic increase in the number of neutrophils entering the brain of WT mice, an effect that was almost totally abolished in the mutant mice and correlated with a significant reduction in the mRNA levels of interleukin-1beta, intracellular adhesion molecule-1 and neutrophil-specific chemokines. These effects were reversed with leptin replenishment in ob/ob mice leading to recovery of neutrophil recruitment into the brain. Moreover, 48 h food deprivation in WT mice, which decreased circulating leptin levels, attenuated the LPS-induced neutrophil recruitment as did a single injection of an anti-leptin antiserum 4 h before LPS treatment in WT mice. These results provide the first demonstration that leptin has a critical role in leukocyte recruitment to the brain following severe systemic inflammation with possible implications for individuals with altered leptin levels such as during obesity or starvation.

The Histone Deacetylase-8 (HDAC8) Selective Inhibitor PCI-34051 Decreases Interleukin-1 Beta Secretion in Vitro and Reduces Inflammation in Vivo

Inhibitors of histone deacetylases (HDACs) which are currently in clinical testing for treating various cancers typically inhibit multiple isoforms of the 11-member HDAC family. We have developed an isoform-selective HDAC inhibitor, PCI-34051, that inhibits HDAC8 with a Ki of 10 nM and greater than 200-fold selectivity over other HDAC isoforms (Balasubramanian et al. (2008) Leukemia,22:1026–34). We have shown that PCI-34051 selectively induced apoptosis in cell lines derived from T-cell lymphomas and leukemias, but not in other tumor or normal cell types. Here we show that it potently inhibits the secretion of the pro-inflammatory cytokine interleukin-1 beta (IL-1b) in lipopolysaccharide (LPS)-stimulated peripheral mononuclear blood cells (PBMC) and isolated monocytes. PCI-34051 inhibited IL-1b secretion (by 80% compared to control) from LPS-stimulated human PBMC with an IC50 of 0.6 uM, which is much lower than the growth inhibitory concentrations of 2.4–4 uM required in T-cell lymphomas. We found that PCI-34051 also inhibited the secretion of interleukin-18 (IL-18) to a similar extent as IL-1b, but secretion of other pro-inflammatory cytokines, including MIP-1b, MCP-1, TNFa, and IL-6, was inhibited to a smaller extent. Interestingly, IL-18, like IL-1b, is synthesized without a signal peptide, and also utilizes the same non-classical endosomal secretory pathway as IL-1b including cleavage of the pro-form by caspase-1. Thus, we theorized that the modulatory effect of PCI-34051 is likely to involve modulation of the post-translational secretory process. In accordance, we found that the IL-1b mRNA levels were reduced by only 20% compared to control, but the intracellular protein levels of the pro-form was increased by >50% in primary monocytes after treatment with PCI-34051, indicating that the mechanism was due to inhibition of the processing from the pro- to the mature form of the cytokine. We showed that this was not due to a direct inhibition of caspase-1 or TACE (TNF-alpha converting enzyme), but is likely due to an as-yet unidentified substrate of HDAC8. In vivo, PCI-34051 inhibited ear swelling induced by oxazolone in a model of contact hypersensitivity in mice, and we showed that this was accompanied by a reduction in IL-1b at both the protein and mRNA levels. Based on this result, we examined the effect of PCI-34051 on IL-1b secretion in human keratinocytes, as well as in PBMC from psoriasis patients, and found that it could reduce IL-1b secretion in both. We found that PCI-34051 decreased IL-1b by 60% in LPS-stimulated PBMC from rheumatoid arthritis (RA) patients, but the pan-HDAC inhibitors which were only weakly inhibitory to HDAC8 did not have this effect, indicating a specific role for HDAC8 in the secretory process. Finally, we found that in unstimulated PBMC from RA patients that had basal production of IL-1b that this could be decreased by 90% by treatment with PCI-34051. Taken together, these findings indicate that PCI-34051 is an active drug that could be useful for the treatment of T-cell lymphoma as well as for autoinflammatory diseases such as RA and psoriasis.

Chronic NMDA Administration Increases Neuroinflammatory Markers in Rat Frontal Cortex: Cross-Talk Between Excitotoxicity and Neuroinflammation

Chronic N-Methyl-D: -aspartate (NMDA) administration, a model of excitotoxicity, and chronic intracerebroventricular lipopolysaccharide infusion, a model of neuroinflammation, are reported to upregulate arachidonic acid incorporation and turnover in rat brain phospholipids as well as enzymes involved in arachidonic acid metabolism. This suggests cross-talk between signaling pathways of excitotoxicity and of neuroinflammation, involving arachidonic acid. To test whether chronic NMDA administrations to rats can upregulate brain markers of neuroinflammation, NMDA (25 mg/kg i.p.) or vehicle (1 ml saline/kg i.p.) was administered daily to adult male rats for 21 days. Protein and mRNA levels of cytokines and other inflammatory markers were measured in the frontal cortex using immunoblot and real-time PCR. Compared with chronic vehicle, chronic NMDA significantly increased protein and mRNA levels of interleukin-1beta, tumor necrosis factor alpha, glial fibrillary acidic protein and inducible nitric oxide synthase. Chronic NMDA receptor overactivation results in increased levels of neuroinflammatory markers in the rat frontal cortex, consistent with cross-talk between excitotoxicity and neuroinflammation. As both processes have been reported in a number of human brain diseases, NMDA receptor inhibitors might be of use in treating neuroinflammation in these diseases.

Role of infection in irritable bowel syndrome

Infection by pathogenic organisms leads to mucosal damage and disruption of the gut's extensive commensal flora, factors which may lead to prolonged bowel dysfunction. Six to 17% of unselected irritable bowel syndrome (IBS) patients believe their symptoms began with an infection, which is supported by prospective studies showing a 4%-31% incidence of postinfectious IBS-(PI) following bacterial gastroenteritis. The wide range of incidence can be accounted for by differences in risk factors, which include in order of magnitude; severity of initial illness > bacterial toxigenicity > hypochondriasis, depression and neuroticism, and adverse life events in the previous 3 months. PI-IBS has been reported after Campylobacter, Salmonella, and Shigella infections. Serial biopsies after Campylobacter jejuni gastroenteritis show an initial inflammatory infiltrate, with an increase in enterochromaffin (EC) cells, which in most cases subsides over the next 6 months. Those who go on to develop IBS show increased numbers of EC and lymphocyte cell counts at 3 months compared with those who do not develop IBS. Interleukin-1beta mRNA levels are increased in the mucosa of those who develop PI-IBS, who also show increased gut permeability. Recover can be slow, with approximately 50% still having symptoms at 5 years. Recent studies suggest an increase in peripheral blood mononuclear cell cytokine production in unselected IBS, an abnormality that may be ameliorated by probiotic treatment. The role of small-bowel bacterial overgrowth in IBS is controversial, but broad-spectrum antibiotics do have a temporary benefit in some patients. More acceptable long-term treatments altering gut flora are awaited with interest.

Cia27 is a novel non-MHC arthritis severity locus on rat chromosome 10 syntenic to the rheumatoid arthritis 17q22–q25 locus

Cia27 on rat chromosome 10 is a collagen-induced arthritis (CIA) severity quantitative trait locus originally identified in a study of (DA x ACI) F2. As an initial step towards the positional cloning of the Cia27 gene, a 17 cM (21 Mb) interval from the DA strain (arthritis-susceptible) containing the two-logarithm of odds support interval comprising Cia27 was introgressed into the ACI (arthritis-resistant) background through genotype-guided congenic breeding. ACI.DA(Cia27) congenics developed a significantly more severe form of arthritis (CIA), with a 5.9-fold increase in median arthritis severity index, a parameter known to correlate with synovial inflammation, and cartilage and bone erosions, compared with ACI (P< or =0.001). The arthritis severity enhancing effect could be detected from day 21 onwards. Rats heterozygous at the congenic interval developed a disease similar to ACI rats, suggesting that DA alleles operate in a recessive manner. Levels of autoantibodies anti-rat type II collagen did not correlate with arthritis severity. Synovial tissue mRNA levels of interleukin-1beta (IL-1beta) were significantly increased in ACI.DA(Cia27) congenics compared with ACI. These results demonstrate that Cia27 harbors a novel arthritis severity regulatory gene. The identification of this gene should facilitate the identification of the rheumatoid arthritis gene mapped to the human syntenic region on chromosome 17q22-q25.

Collagen degradation in a murine myocarditis model: relevance of matrix metalloproteinase in association with inflammatory induction.

Myocardial collagen degradation is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinase (TIMPs). The possible relevance of MMPs in association with the inflammatory induction was investigated in a murine coxsackievirus B3 myocarditis model. Hearts from viral infected and sham-infected BALB/c mice were analyzed by semi-quantitative RT-PCR, picrosirius red staining, Western blot analysis, and immunohistochemistry. In viral infected mice, both mRNA and protein abundance for collagen type I remained unaltered. In addition, picrosirius red staining showed the unchanged total collagen content. However, degraded soluble fraction of collagen type I protein was increased. Moreover, the mRNA abundance for MMP-3 and MMP-9 was upregulated, whereas the mRNAs for TIMP-1 and TIMP-4 were downregulated, respectively. The upregulation of MMP-3/MMP-9 and downregulation of TIMP-4 were confirmed at the protein level, and were associated with significantly increased mRNA levels of interleukin 1beta, tumor necrosis factor-alpha, transforming growth factor-beta1 and interleukin-4. The increment of MMPs in the absence of counterbalance by TIMPs may lead to a functional defect of the myocardial collagen network by posttranslational mechanisms. This may contribute significantly to the development of left ventricular dysfunction in murine viral myocarditis. The inflammatory response with induction of cytokines may mediate the dysregulation of the myocardial MMP/TIMP systems.

Isolation of three high molecular weight polysaccharide preparations with potent immunostimulatory activity from Spirulina platensis, aphanizomenon flos-aquae and Chlorella pyrenoidosa.

This research describes the identification of three new high molecular weight polysaccharide preparations isolated from food-grade microalgae that are potent activators of human monocytes/macrophages: "Immulina" from Spirulina platensis, "Immunon" from Aphanizomenon flos-aquae, and "Immurella" from Chlorella pyrenoidosa. These polysaccharides are structurally complex and have estimated molecular weights above ten million daltons. All three polysaccharides are highly water soluble and comprise between 0.5 % and 2.0 % of microalgal dry weight. Immunostimulatory activity was measured using a transcription factor-based bioassay for nuclear factor kappa B (NF-kappa B) activation in THP-1 human monocytes/macrophages. Using this system the EC(50) values for these microalgal polysaccharides are between 20 and 110 ng/ml (about 10pM). THP-1 activation was confirmed by measuring immune cytokine mRNA induction using reverse transcriptase-polymerase chain reaction (RT-PCR). Each polysaccharide substantially increased mRNA levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). These polysaccharides are between one hundred and one thousand times more active for in vitro monocyte activation than polysaccharide preparations that are currently used clinically for cancer immunotherapy.

Attenuation of colon inflammation through activators of the retinoid X receptor (RXR)/peroxisome proliferator-activated receptor gamma (PPARgamma) heterodimer. A basis for new therapeutic strategies.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is highly expressed in the colon mucosa and its activation has been reported to protect against colitis. We studied the involvement of PPARgamma and its heterodimeric partner, the retinoid X receptor (RXR) in intestinal inflammatory responses. PPARgamma(1/)- and RXRalpha(1/)- mice both displayed a significantly enhanced susceptibility to 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis compared with their wild-type littermates. A role for the RXR/PPARgamma heterodimer in the protection against colon inflammation was explored by the use of selective RXR and PPARgamma agonists. TNBS-induced colitis was significantly reduced by the administration of both PPARgamma and RXR agonists. This beneficial effect was reflected by increased survival rates, an improvement of macroscopic and histologic scores, a decrease in tumor necrosis factor alpha and interleukin 1beta mRNA levels, a diminished myeloperoxidase concentration, and reduction of nuclear factor kappaB DNA binding activity, c-Jun NH(2)-terminal kinase, and p38 activities in the colon. When coadministered, a significant synergistic effect of PPARgamma and RXR ligands was observed. In combination, these data demonstrate that activation of the RXR/PPARgamma heterodimer protects against colon inflammation and suggest that combination therapy with both RXR and PPARgamma ligands might hold promise in the clinic due to their synergistic effects.

Effects of Interleukin-10 (IL-10) on Pain Behavior and Gene Expression Following Excitotoxic Spinal Cord Injury in the Rat

Intraspinal injection of quisqualic acid (QUIS) produces excitotoxic injury with pathophysiological characteristics similar to those associated with ischemic and traumatic spinal cord injury (SCI). Responses to QUIS-induced injury include an inflammatory component, as well as the development of spontaneous and evoked pain behaviors. We hypothesized that QUIS-induced inflammation and subsequent gene expression contribute to the development and progression of pain-related behaviors and that blockade of inflammation-related gene expression leads to the amelioration of these behaviors. Using the QUIS model of spinal cord injury, we examined whether interleukin-10 (IL-10), a potent anti-inflammatory cytokine, is able to reduce mRNA levels of inflammatory and cell death-related genes leading to a reduction of pain behaviors. The results demonstrate that animals receiving systemic injection of IL-10, 30 minutes following QUIS-induced SCI, showed a significant delay in the onset of excessive grooming behavior, a significant reduction in grooming severity, and a significant reduction in the longitudinal extent of a pattern of neuronal loss within the spinal cord characterized as “grooming-type damage.” QUIS injections also resulted in an increase in mRNA levels of interleukin-1 beta (IL-1β), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), CD95 ligand (CD95-L, also called FAS-L/APO-1L), and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Results of QUIS injury plus IL-10 treatment resulted in a significant downregulation of IL1-β and iNOS mRNA and these results were supported by Western blot analysis of protein levels following IL-10 treatment. These data suggest that IL-10 reduces inflammation and that targeting injury-induced inflammation is an effective strategy for limiting the extent of neuronal damage following excitotoxic SCI and thus the onset and progression of injury-induced pain behaviors.

Characterization of human monocyte activation by a water soluble preparation of Aphanizomenon flos-aquae.

Aphanizomenon flos-aquae (AFA) is a fresh-water microalgae that is consumed as a nutrient-dense food source and for its health-enhancing properties. The current research characterizes the effect of a water soluble preparation from AFA on human monocyte/macrophage function and compares the effect of AFA with responses from known agents that modulate the immune system. At 0.5 pg/ml the AFA extract robustly activated nuclear factor kappa B (NF-kappa B) directed luciferase expression in THP-1 human monocytic cells to levels at 50% of those achieved by maximal concentrations (10 microg/ml) of bacterial lipopolysaccharide (LPS). In addition, the AFA extract substantially increased mRNA levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), and enhanced the DNA binding activity of NF-kappa B. The effects of AFA water soluble preparation were similar to the responses displayed by LPS, but clearly different from responses exhibited by tetradecanoyl phorbol acetate (TPA) and interferon-gamma (INF-gamma). Pretreatment of THP-1 monocytes with factors known to induce hyporesponsiveness suppressed both AFA-dependent and LPS-dependent activation. These results suggest that the macrophage-activating properties of the AFA water soluble preparation are mediated through pathways that are similar to LPS-dependent activation.

Activation of inflammatory mediators in rat renal isografts by donor brain death.

Brain death (BD) has been thought to influence the early course of transplanted organs by triggering a series of nonspecific inflammatory events that in turn may increase the kinetics and intensity of the immunological host responses. In this study early nonspecific, cellular, and molecular changes occurring in kidney isografts from BD donors are compared with those from normal anesthetized, ventilated controls. After induction of brain death, the animals were mechanically ventilated for 6 hr before organ removal. Only rats with stable blood pressure (mean arterial pressure >80 mmHg) were included. Serum creatinines were measured daily. Representative grafts were harvested 6 hr after brain death and between 1 hr and 5 days after engraftment for morphology, immunohistology, and reverse transcriptase-polymerase chain reaction. The presence of serum cytokines was assessed by enzyme linked immunoabsorbant assay. Serum creatinine levels rose slightly in recipients from BD donors. Serum interleukin-1beta levels increased within 6 hr versus controls (P<0.05). mRNA levels of interleukin-1beta and macrophage inhibitory protein-1 in the kidneys were up-regulated transiently before engraftment (6 hr after BD) and 1 hr after revascularization (P<0.05). By immunohistology, numbers of infiltrating polymorphonuclear leukocytes peaked at 24 hr in parallel with intragraft induction of P- and E-selectin, complement, and other proinflammatory chemokines and cytokines. At 5 days, the isografts from BD donors were highly infiltrated by host leukocyte populations associated with intense up-regulation of their products. In contrast, those from control donors remained relatively normal through this initial follow-up period. The intense nonimmune inflammation produced in isografts after donor BD may represent the initial stages of a continuum between an initial nonspecific and later immune reactivity, when placed in the context of allotransplantation.

Association of the course of collagen-induced arthritis with distinct patterns of cytokine and chemokine messenger RNA expression.

To quantitate changes in cytokine and chemokine messenger RNA (mRNA) levels during the development and progression of collagen-induced arthritis (CIA) in mice. Mice with CIA were scored for arthritis and killed at weekly intervals. Cytokine and chemokine mRNA levels were determined by RNase protection assays of total paw RNA. Arthritic paws exhibited mRNA levels of interleukin-1beta (IL-1beta), IL-2, macrophage inflammatory protein 2 (MIP-2), IL-6, IL-1 receptor antagonist, RANTES, tumor necrosis factor alpha (TNFalpha), TNFbeta, MIP-1alpha, IL-11, transforming growth factor beta1 (TGFbeta1), TGFbeta2, and TGFbeta3 that were increased above mRNA levels in paws of normal, unimmunized mice and that exhibited distinct temporal patterns of mRNA expression. Clinically uninvolved paws also exhibited an increase in mRNA levels of IL-11, RANTES, TNFalpha, TNFbeta, and MIP-1alpha. The observed differential temporal cytokine and chemokine mRNA expression patterns suggest that specific cytokines and chemokines have defined roles at various times during the course of autoimmune arthritis. Since most of these cytokines and chemokines are found in human rheumatoid arthritis (RA) synovium and synovial fluids, these findings may have relevance to RA.

Effects of ethanol on gene expression in rat bone: transient dose-dependent changes in mRNA levels for matrix proteins, skeletal growth factors, and cytokines are followed by reductions in bone formation.

Several studies were performed in female rats to determine dose and time course changes in mRNA levels for matrix proteins in bone after a single administration of ethanol. As expected, dose-dependent transient increases in blood ethanol were measured. Additionally, there was mild hypocalcemia with no change in immunoreactive parathyroid hormone. Coordinated dose-dependent increases in mRNA for type 1 collagen, osteonectin, and osteocalcin were noted in the proximal tibial metaphysis 6 hr after ethanol was given, with the peak values occurring at a dose of 1.2 g/kg (0.4 ml). Similar increases in mRNA levels for matrix proteins were noted in lumbar vertebrae after ethanol treatment. The changes were specific for bone; ethanol had no effect on mRNA levels for matrix proteins in the uterus or liver, although the mRNA concentrations tended to be reduced in uterus. Message levels for several cytokines implicated in the regulation of bone turnover were also assayed; mRNA levels for transforming growth factor-beta1, transforming growth factor-beta2, interferon-gamma, and interleukin-6 were unchanged at doses ranging from 0.14 to 1.7 g/kg. At the highest dose of ethanol, the mRNA level for tumor necrosis factor-alpha was elevated while the level for insulin-like growth factor-1 was reduced. The time course effects of ethanol (0.4 ml dose) were determined in a separate experiment. Ethanol resulted in a transient increase in mRNA levels for the three bone matrix proteins assayed. However, matrix protein synthesis, as determined by incorporation of 3H-proline into the proximal tibial metaphysis, was not changed after 6 hr. The changes in mRNA levels for the matrix proteins were preceded by brief, transient decreases in mRNA levels for interleukin-1beta, interferon-gamma, and migration inhibitory factor, and followed by a more prolonged decrease in the mRNA level for insulin-like growth factor-1. A subsequent study was performed to determine the effects of repetitive daily treatment with ethanol on rat bone. After 7 days, there were highly significant decreases in the mRNA level for type 1 collagen, as well as decreased bone formation. These results suggest that ethanol may alter bone metabolism by disturbing signal transduction pathways that regulate the expression of genes for bone matrix proteins, skeletal growth factors, and cytokines.

Interleukin 1 receptor antagonist gene expression in rat pituitary in the systemic inflammatory response syndrome: pathophysiological implications.

The systemic inflammatory response syndrome (SIRS) is characterized by endothelial cell activation causing a generalized inflammatory response and cytokine-mediated pathophysiological alterations. The pathophysiology of SIRS involves changes in the functioning of several endocrine glands, including the pituitary. SIRS-induced alterations in pituitary function include activation of the hypothalamic-pituitary adrenal (HPA) axis causing hypercortisolemia, the euthyroid sick syndrome, and cessation of reproductive function. These changes are in part mediated by cytokines, such as tumor necrosis factor alpha (TNF-alpha) and interleukin 1 (IL-1). IL-1 function depends on the local ratio of bioactive IL-1 and IL-1 receptor antagonist. Here we review our studies on IL-1 beta and IL-1ra mRNA levels in the pituitary in a rat model of SIRS. We have found that IL-1 beta mRNA peaked at 2 h after lipopolysaccharide (LPS) administration, increasing twelvefold over control values in the posterior pituitary and fivefold over controls in the anterior pituitary. IL-1ra mRNA levels peaked at 6 h post-LPS administration, later than those of IL-1 beta mRNA. IL-1ra mRNA levels increased tenfold in the anterior pituitary, but were induced only threefold in the posterior pituitary. IL-1ra gene expression is profoundly induced in the pituitary in vivo during systemic inflammation and its induction follows that of IL-1 beta, but it is differentially regulated and tissue-specific, occurring predominantly in the anterior pituitary. Future studies of the effects of IL-1 in the pituitary should take into account the local levels of IL-1ra.