Infrared radiation from cage bedding moderates rat inflammatory and autoimmune responses in collagen-induced arthritis.

The development of collagen type II (CII)-induced arthritis (CIA), a model of rheumatoid arthritis, in rats housed in cages with bedding composed of Celliant fibres containing ceramic particles, which absorb body heat and re-emit the energy back to the body in the form of infrared radiation (+IRF rats), and those housed in cages with standard wooden shaving bedding (-IRF control rats) was examined. The appearance of the first signs of CIA was postponed, while the disease was milder (judging by the arthritic score, paw volume, and burrowing behaviour) in +IRF compared with -IRF rats. This correlated with a lower magnitude of serum anti-CII IgG antibody levels in +IRF rats, and lower production level of IL-17, the Th17 signature cytokine, in cultures of their paws. This could be partly ascribed to impaired migration of antigen-loaded CD11b + dendritic cells and their positioning within lymph nodes in +IRF rats reflecting diminished lymph node expression of CCL19 /CCL21. Additionally, as confirmed in rats with carrageenan-induced paw inflammation (CIPI), the infrared radiation from Celliant fibres, independently from immunomodulatory effects, exerted anti-inflammatory effects (judging by a shift in pro-inflammatory mediator to anti-inflammatory/immunoregulatory mediator ratio towards the latter in paw cultures) and ameliorated burrowing behaviour in CIA rats.

Molecular basis of carrageenan-induced cytokines production in macrophages.

BACKGROUND: Low molecular weight carrageenan (Cg) is a seaweed-derived sulfated polysaccharide widely used as inflammatory stimulus in preclinical studies. However, the molecular mechanisms of Cg-induced inflammation are not fully elucidated. The present study aimed to investigate the molecular basis involved in Cg-induced macrophages activation and cytokines production. METHODS: Primary culture of mouse peritoneal macrophages were stimulated with Kappa Cg. The supernatant and cell lysate were used for ELISA, western blotting, immunofluorescence. Cg-induced mouse colitis was also developed. RESULTS: Here we show that Cg activates peritoneal macrophages to produce pro-inflammatory cytokines such as TNF and IL-1ß. While Cg-induced TNF production/secretion depends on TLR4/MyD88 signaling, the production of pro-IL-1ß relies on TLR4/TRIF/SYK/reactive oxygen species (ROS) signaling pathway. The maturation of pro-IL1ß into IL-1ß is dependent on canonical NLRP3 inflammasome activation via Pannexin-1/P2X7/K+ efflux signaling. In vivo, Cg-induced colitis was reduced in mice in the absence of NLRP3 inflammasome components. CONCLUSIONS: In conclusion, we unravel a critical role of the NLRP3 inflammasome in Cg-induced pro-inflammatory cytokines production and colitis, which is an important discovery on the pro-inflammatory properties of this sulfated polysaccharide for pre-clinical studies. Video abstract Carrageenan (Cg) is one the most used flogistic stimulus in preclinical studies. Nevertheless, the molecular basis of Cg-induced inflammation is not totally elucidated. Herein, Lopes et al. unraveled the molecular basis for Cg-induced macrophages production of biological active IL-1ß. The Cg-stimulated macrophages produces pro-IL-1ß depends on TLR4/TRIF/Syk/ROS, whereas its processing into mature IL-1ß is dependent on the canonical NLRP3 inflammasome.

Local and Systemic Profiles of Inflammatory Cytokines in Carrageenan-induced Paw Inflammation in Rats.

OBJECTIVE: Carrageenan (CA)-induced edema has been described as highly reproducible model of acute inflammation. However, little is known about the cytokines attributed to the CA-induced inflammation. In this study, we aimed to investigate the local and systemic expression profiles of various inflammatory cytokines following the subplantar injection of CA in rats. METHODOLOGY: Acute inflammation was induced in male Wistar rats by subplantar injection of CA. Serum and paw tissue were examined for the level of 19 specific inflammatory cytokines using antibody array. Further, the CA-elicited level of key inflammatory cytokines, cytokine-induced neutrophil chemoattractant (CINC)-2, CINC-3, interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α, were quantified by enzyme-linked immunosorbent assay (ELISA). RESULTS: Edema was peaked 3 h postinjection of CA in hind paw. Among 19 specific cytokines profiled using antibody array, CA significantly (p < 0.05) elicited the levels of CINC-2, CINC-3, IL-1ß, IL-6, ß-NGF, TNF-α, and VEGF in paw tissue and that of CINC-2 and CINC-3 in serum. Consistently, levels of CINC-2, CINC-3, IL-1ß, IL-6, and TNF-α in tissue and CINC-2 and CINC-3 in serum were upregulated in CA-treated rats when compared to control, quantified by ELISA. CONCLUSIONS: This study corroborates the distinct pattern of inflammatory cytokines involved during CA-induced acute inflammation. Furthermore, data provide new evidence on elevated expression of rat CXC chemokines: CINC-2 and CINC-3 at the site of inflammation as well as their significant reflection in the circulation, thereby suggesting their frontline role in CA-induced acute inflammation.

Enhanced effect of κ-carrageenan on TNBS-induced inflammation in mice.

BACKGROUND: As a sulfated polysaccharide, carrageenan has been widely used as common food additive. METHODS: In the present study, we investigated the effects of κ-carrageenan on TNBS-induced gut inflammation in mice. BALB/c mice were pretreated with κ-carrageenan for 14days prior to the administration of TNBS. RESULTS: Our results showed that κ-carrageenan pretreatment aggravated the loss of body weight and further increased the mortality rate. Histological and morphological analyses revealed that the TNBS-induced colonic inflammation was deteriorated by the κ-carrageenan administration. The ratio of CD4(+)CD25(+)CD127dim/CD4(+) of the κ-carrageenan+TNBS groups was significantly lower than that of the TNBS group. The expression of IL-2, TNF-α and IL-6 was significantly increased, whereas the expression of IL-10 was significantly decreased in the κ-carrageenan+TNBS groups. In addition, κ-carrageenan, together with TNBS, decreased the enzyme activity of SOD and GSH-px and up-regulated the expression of TLR4, NF-κB, p-ERK, p-JNK, p-Jun., IL-8 and MDA in the colonic mucosa. CONCLUSIONS: κ-Carrageenan aggravated the TNBS-induced intestinal inflammation, and such an effect could be associated with the oxidative stress and activation of TLR4-NF-κB and MAPK/ERK1/2 pathway.

Riparin A, a compound from Aniba riparia, attenuate the inflammatory response by modulation of neutrophil migration.

Inflammation is a local tissue response to attacks characterized by vascular and cellular events, including intense oxidative stress. Riparin A, a compound obtained from Aniba riparia, has been shown to have antioxidant activity and cytotoxicity in vitro. This study was aimed at evaluating the anti-inflammatory effect of riparin A against acute inflammation. The results of our evaluations in various experimental models indicated that riparin A reduced paw edema induced by carrageenan, compound 48/80, histamine, and serotonin. Furthermore, it decreased leukocyte and neutrophil counts, myeloperoxidase activity, thiobarbituric acid reactive substance (TBARS) levels, and cytokine (tumor necrosis factor-α and interleukin-1ß) levels increased by carrageenan-induced peritonitis, and reversed glutathione levels. Riparin A also reduced carrageenan-induced adhesion and rolling of leukocytes on epithelial cells and did not produce gastric-damage as compared with indomethacin. In conclusion, the data show that riparin A reduces inflammatory response by inhibiting vascular and cellular events, modulating neutrophil migration, inhibiting proinflammatory cytokine production, and reducing oxidative stress.

Anti-inflammatory and anti-tumor activity of the marine mangrove Rhizophora apiculata.

A methanolic extract of Rhizophora apiculata was evaluated for its anti-inflammatory and anti-tumor activity against B16F10 melanoma cells in BALB/c mice. The administration of R. apiculata extract was shown to inhibit the solid tumor development in mice. R. apiculata treatment significantly reduced tumor cell glutathione (GSH) levels as well as serum γ-glutamyl transpeptidase (GGT) and nitric oxide (NO) levels in the tumor-bearing animals. The total white blood cell count and hemoglobin levels were also significantly increased in extract-treated hosts. The use of R. apiculata substantially reduced the acute inflammation (assessed as paw edema) induced by carrageenan and also reduced inflammation edema induced by formalin. Analysis of this methanolic extract revealed a high content of 4-pyrrolidinyl, pyrazole, and ketone derivatives. These studies suggest that R. apiculata extract could be used as a (natural) anti-inflammatory and anti-tumor agent.

Inhibition of pro-inflammatory mediators: role of Bacopa monniera (L.) Wettst.

Bacopa monniera (L.) Wettst is a renowned plant in the Ayurvedic system of medicine. The present study seeks to identify the anti-inflammatory activity of two fractions from the methanolic extract of Bacopa, viz. the triterpenoid and bacoside-enriched fractions. The ability of these two fractions to inhibit the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 was tested using lipopolysaccharide (LPS)-activated peripheral blood mononuclear cells and peritoneal exudate cells in vitro. We found that triterpenoid and bacoside-enriched fractions significantly inhibited LPS-activated TNF-α, IL-6 and nitrite production in mononuclear cells. Significant antioxidant activity was exhibited by the bacoside enriched fraction compared to the triterpenoid fraction. Carrageenan-induced hind paw oedema assay revealed that triterpenoid and bacoside-enriched fractions exerted anti-oedematogenic effect, while in the arthritis model only the triterpenoid fraction exerted an anti-arthritic potential. The present study provides an insight into the ability of Bacopa monniera to inhibit inflammation through modulation of pro-inflammatory mediator release.

Anti-inflammatory effects of black rice, cyanidin-3-O-beta-D-glycoside, and its metabolites, cyanidin and protocatechuic acid.

The anti-inflammatory effects of cyanidin-3-O-beta-D-glycoside (C3G), a major constituent of black rice (BR), and its metabolites, cyanidin and protocatechuic acid (PA), were assessed in lipopolysaccharide (LPS)-induced RAW 264.7 cells and carrageenan-induced inflammation in air pouches in BALB/c mice. BR, C3G and its metabolites suppressed the production of the proinflammatory cytokines, TNF-alpha and IL-1 beta, and the inflammatory mediators, NO and prostaglandin E2 (PGE2), as well as the gene expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in RAW 264.7 cells. These agents also inhibited the phosphorylation of I kappaB-alpha, the nuclear translocation of NF-kappaB, and the activation of mitogen-activated protein kinases. Furthermore, these agents significantly inhibited the leukocyte number and the levels of TNF-alpha, PGE2, and protein in the exudates of the air pouch in carrageenan-treated mice, as well as COX-2 expression and NF-kappaB activation. Among the test agents, PA most potently inhibited these inflammatory mediators in vivo and in vitro. Based on these findings, if BR is orally administered, its main constituent, C3G, may be metabolized to cyanidin and/or PA, which express potent anti-inflammatory effects by regulating NF-kappaB and MAPK activation.

Carrageenan as an adjuvant to enhance peptide-based vaccine potency.

New innovative therapies are urgently required in order to combat the high mortality and morbidity associated with advanced cancers. Antigen-specific cancer immunotherapy using peptide-based vaccination has emerged as an attractive approach for the control of cancers due to its simplicity and easy preparation. However, such an approach requires the employment of suitable adjuvants. In the current study, we explored the employment of a sulfated polysaccharide compound from red algae, carrageenan (CGN) as an adjuvant for their ability to generate antigen-specific immune responses and antitumor effects in mice vaccinated with human papillomavirus type 16 (HPV-16) E7 peptide vaccine. We found that carrageenan can significantly enhance the E7-specific immune responses generated by E7 peptide vaccination via the TLR4 activation pathway. In addition, carrageenan could enhance the protective and therapeutic antitumor effects generated by E7 peptide vaccination against E7-expressing tumors. Furthermore, the observed enhancement was not restricted to E7 antigen but was also applicable to other antigenic systems. We also found that other structurally similar compounds to CGN, such as dextran, also generated similar immune enhancement. Thus, our data suggest that CGN and its structurally related compounds may serve as innovative adjuvants for enhancing peptide-based vaccine potency.

P2X4 receptors mediate PGE2 release by tissue-resident macrophages and initiate inflammatory pain.

Prostaglandin E2 (PGE2) is a key mediator of inflammation and contributes to pain hypersensitivity by promoting sensory neurons hyperexcitability. PGE2 synthesis results from activation of a multi-step enzymatic cascade that includes cyclooxygenases (COXs), the main targets of non-steroidal anti-inflammatory drugs (NSAIDs). Although NSAIDs are widely prescribed to reduce inflammatory symptoms such as swelling and pain, associated harmful side effects restrict their long-term use. Therefore, finding new drugs that limit PG production represents an important therapeutic issue. In response to peripheral inflammatory challenges, mice lacking the ATP-gated P2X4 channel (P2X4R) do not develop pain hypersensitivity and show a complete absence of inflammatory PGE2 in tissue exudates. In resting conditions, tissue-resident macrophages constitutively express P2X4R. Stimulating P2X4R in macrophages triggers calcium influx and p38 MAPK phosphorylation, resulting in cytosolic PLA2 (cPLA2) activation and COX-dependent release of PGE2. In naive animals, pain hypersensitivity was elicited by transfer into the paw of ATP-primed macrophages from wild type, but not P2X4R-deficient mice. Thus, P2X4Rs are specifically involved in inflammatory-mediated PGE2 production and might therefore represent useful therapeutic targets.

Tumor bearing decreases systemic acute inflammation in rats--role of mast cell degranulation.

OBJECTIVE AND DESIGN: To investigate the effect of experimental tumor bearing on acute inflammation models in rats. METHODS: Four and 7 days after Walker tumor implantation in the right armpit, carrageenan or dextran- induced edema in the contralateral paw, carrageenan induced neutrophil migration into peritoneal cavities, cutaneous vascular permeability induced by bradykinin, histamine, serotonin, substance P, capsaicin or compound 48/80, and mesenteric mast cell degranulation induced by compound 48/80 were evaluated. The control group did not receive tumor implantation. Statistical analysis was performed using one way analysis of variance (ANOVA) followed by the Bonferroni test. RESULTS: On the 7(th) day after tumor inoculation, there were significant decreases in both carrageenan and dextran- induced paw edema. Tumor bearing did not change the neutrophil infiltration induced by carrageenan. There were decreases in cutaneous vascular permeability induced by compound 48/80, serotonin or bradykinin, but not that induced by histamine, substance P. A significant inhibition of mesenteric mast cell degranulation induced by compound 48/80 was observed, on the 4(th) and 7(th) days after tumor inoculation. CONCLUSION: Tumor bearing can limit mast cell function and vascular events in acute systemic inflammation in rats, without changes in neutrophil migration.

DMBT1 functions as pattern-recognition molecule for poly-sulfated and poly-phosphorylated ligands.

Deleted in malignant brain tumors 1 (DMBT1) is a secreted glycoprotein displaying a broad bacterial-binding spectrum. Recent functional and genetic studies linked DMBT1 to the suppression of LPS-induced TLR4-mediated NF-kappaB activation and to the pathogenesis of Crohn's disease. Here, we aimed at unraveling the molecular basis of its function in mucosal protection and of its broad pathogen-binding specificity. We report that DMBT1 directly interacts with dextran sulfate sodium (DSS) and carrageenan, a structurally similar sulfated polysaccharide, which is used as a texturizer and thickener in human dietary products. However, binding of DMBT1 does not reduce the cytotoxic effects of these agents to intestinal epithelial cells in vitro. DSS and carrageenan compete for DMBT1-mediated bacterial aggregation via interaction with its bacterial-recognition motif. Competition and ELISA studies identify poly-sulfated and poly-phosphorylated structures as ligands for this recognition motif, such as heparansulfate, LPS, and lipoteichoic acid. Dose-response studies in Dmbt1(-/-) and Dmbt1(+/+) mice utilizing the DSS-induced colitis model demonstrate a differential response only to low but not to high DSS doses. We propose that DMBT1 functions as pattern-recognition molecule for poly-sulfated and poly-phosphorylated ligands providing a molecular basis for its broad bacterial-binding specificity and its inhibitory effects on LPS-induced TLR4-mediated NF-kappaB activation.

Inhibitory effect of amygdalin on lipopolysaccharide-inducible TNF-alpha and IL-1beta mRNA expression and carrageenan-induced rat arthritis.

Amygdalin is a cyanogenic glycoside plant compound found in the seeds of rosaceous stone fruits. We evaluated the antiinflammatory and analgesic activities of amygdalin, using an in vitro lipopolysaccharide (LPS)-induced cell line and a rat model with carrageenan-induced ankle arthritis. One mM amygdalin significantly inhibited the expression of TNF-alpha and IL-1beta mRNAs in LPS-treated RAW 264.7 cells. Amygdalin (0.005, 0.05, and 0.1 mg/kg) was intramuscularly injected immediately after the induction of carrageenan-induced arthritic pain in rats, and the anti-arthritic effect of amygdalin was assessed by measuring the weight distribution ratio of the bearing forces of both feet and the ankle circumference, and by analyzing the expression levels of three molecular markers of pain and inflammation (c-Fos, TNF-alpha, and IL-1beta) in the spinal cord. The hyperalgesia of the arthritic ankle was alleviated most significantly by the injection of 0.005 mg/kg amygdalin. At this dosage, the expressions of c-Fos, TNF-alpha, and IL-1beta in the spinal cord were significantly inhibited. However, at dosage greater than 0.005 mg/kg, the painrelieving effect of amygdalin was not observed. Thus, amygdalin treatment effectively alleviated responses to LPStreatment in RAW 264.7 cells and carrageenan-induced arthritis in rats, and may serve as an analgesic for relieving inflammatory pain.

Anti-inflammatory activity of an orange peel polymethoxylated flavone, 3',4',3,5,6,7,8-heptamethoxyflavone, in the rat carrageenan/paw edema and mouse lipopolysaccharide-challenge assays.

The anti-inflammatory properties of 3',4',3,5,6,7,8-heptamethoxyflavone (HMF), a citrus polymethoxylated flavone, were studied in the bacterial lipopolysaccharide (LPS)-challenge/tumor necrosis factor-alpha (TNFalpha) response in mice and in the carrageenan/paw edema assay in rats. In each of these trials, HMF administered by intraperitoneal (ip) injection exhibited anti-inflammatory activity, whereas HMF administered orally (po) produced no effects. The inhibition observed in the LPS-challenge/TNFalpha assay correlated with the HMF levels in the blood sera of mice dosed (ip) with either 33 or 100 mg/kg body weight. Low levels of HMF (0.035 +/- 0.024 ppm) were detected in the blood sera of mice dosed orally [100 mg of HMF (suspended in vegetable oil)/kg], whereas ip injection led to higher levels (0.517 +/- 0.051 ppm). This may account for the different levels of anti-inflammatory effects observed in mice following ip vs oral HMF administration. HMF metabolites, including a number of mono- and di-demethylated HMF metabolites and their glucuronic acid conjugates, were also detected, but results of these studies suggest that the glucuronidated metabolites of HMF are inactive in these inflammation models.

Magnetically enriched bone marrow-derived macrophages loaded in vitro with iron oxide can migrate to inflammation sites in mice.

In vitro labelling of cells permits incorporation of large amounts of iron oxide and consequently high detection sensitivity, but it remains controversial whether labelled cells would respond normally to stimuli. This question was addressed by differentiating bone marrow-derived macrophages (BMDMs) in vitro, labelling cells with high concentrations of Endorem in vitro, and eliminating unlabelled cells by magnetic enrichment. To explore their acute inflammatory response, enriched cells were injected into mice with carrageenan-induced inflammation, the 'air pouch model'. Cells recovered from the inflammation site 16 h after intravenous BMDM injection into the tail vein were analysed by in vitro MRI and fluorescent microscopy. With both assays, Endorem-labelled cells were detectable. This indicates that BMDMs, loaded with high concentrations of iron oxide in vitro, can still respond to chemokine gradients and infiltrate inflamed tissue in mice. Furthermore, by using genetically modified mice as BMDM donors, it should be possible to study the role of individual genes in macrophage recruitment.

Kruppel-like factor 2 (KLF2) regulates proinflammatory activation of monocytes.

The mechanisms regulating activation of monocytes remain incompletely understood. Herein we provide evidence that Kruppel-like factor 2 (KLF2) inhibits proinflammatory activation of monocytes. In vitro, KLF2 expression in monocytes is reduced by cytokine activation or differentiation. Consistent with this observation, KLF2 expression in circulating monocytes is reduced in patients with chronic inflammatory conditions such as coronary artery disease. Adenoviral overexpression of KLF2 inhibits the LPS-mediated induction of proinflammatory factors, cytokines, and chemokines and reduces phagocytosis. Conversely, short interfering RNA-mediated reduction in KLF2 increased inflammatory gene expression. Reconstitution of immunodeficient mice with KLF2-overexpressing monocytes significantly reduced carrageenan-induced acute paw edema formation. Mechanistically, KLF2 inhibits the transcriptional activity of both NF-kappaB and activator protein 1, in part by means of recruitment of transcriptional coactivator p300/CBP-associated factor. These observations identify KLF2 as a novel negative regulator of monocytic activation.

Superoxide-related signaling cascade mediates nuclear factor-kappaB activation in acute inflammation.

The nuclear factor-kappaB (NF-kappaB) is a transcription factor that plays a pivotal role in the induction of genes involved in physiological processes, as well as in the response to inflammation. In this study, we used a selective nonpeptidyl superoxide dismutase mimetic, M40403, to investigate the role of superoxide anion in NF-kappaB activation during acute inflammation in mice. Injection of carrageenan into the pleural cavity of mice induced an acute inflammatory response characterized by fluid accumulation in the pleural cavity that contained a large number of neutrophils, as well as an increased production of tumor necrosis factor-alpha and interleukin-1beta. All parameters of inflammation were attenuated by M40403 (10 mg/kg i. p., 30 min prior to carrageenan administration). These inflammatory events were associated with the activation of NF-kappaB in the lung. In particular, the appearance of inhibitory protein kappaB-alpha (IkappaB-alpha) in homogenates of lung tissues was investigated by immunoblot analysis at 4 h after carrageenan administration. IkappaB-alpha levels were substantially reduced in the lung tissue from carrageenan-treated mice in comparison with sham-treated mice. Furthermore, to detect NF-kappaB/DNA binding activity, whole extracts from lung tissue of each mouse were analyzed by electrophoretic mobility-shift assay. The DNA binding activity significantly increased in whole extracts obtained from lung tissues of vehicle-treated mice 4 h after carrageenan administration. Treatment of mice with M40403 caused a significant inhibition of carrageenan-induced IkappaB-alpha degradation and NF-kappaB/DNA binding activity. These data confirm that M40403 exerts a potent antiinflammatory activity and clearly demonstrate that the reduction of the inflammatory process is associated with modification of the activation of signal transduction pathways.

The role of macrophages in the induction of murine immune response to Actinobacillus actinomycetemcomitans.

The aim of this study was to determine the role of macrophages in the Actinobacillus actinomycetemcomitans-induced murine immune response. BALB/c mice were given carrageenan solution by intraperitoneal injection before immunisation with heat-killed A. actinomycetemcomitans. Mice immunised with antigens and phosphate-buffered saline served as positive and negative controls, respectively. One week after the last immunisation, the delayed-type hypersensitivity (DTH) response was assessed by measurement of footpad swelling. Serum IgG and IgM anti-A. actinomycetemcomitans antibody levels and culture supernate levels of interferon (IFN)-gamma were determined by ELISA. The diameter of abscess formation was determined every 5 days. Sham-immunised spleen cells were transferred to carrageenan-untreated recipients (groups A and B) and to carrageenan-treated recipients (group D). Antigen-immunised spleen cells were transferred to carrageenan-untreated (group C) and carrageenan-treated (group E) recipients. The carrageenan-treated recipients in groups F and G received macrophages from antigen- and sham-immunised mice respectively. All mice except those in group A were immunised with antigen 24 h after cell transfer. After 1 week, a partial suppression of DTH response, reduced levels of IFN-gamma, serum IgG and IgM anti-A. actinomycetemcomitans antibodies and delayed healing were seen in carrageenan-treated mice when compared with the positive control. The immune response to A. actinomycetemcomitans in groups A, B and D was lower than that in groups C and E. Healing of the lesion in the former groups was also delayed when compared with the latter groups. The immune response and the healing of the lesion could be partially restored in carrageenan-treated mice that received antigen-pulsed macrophages (group F) but not in those that received naive macrophages (group G). These results suggest that macrophages play a partial role in the induction of the murine immune response to A. actinomycetemcomitans.

The effects of IL-6 and IL-10 and their specific antibodies in the acute inflammatory responses induced by carrageenan in the mouse model of pleurisy.

This study evaluates the effects of intrapleural (i.pl.) injection of interleukin (IL-6) and IL-10 and their specific antibodies on the early (4 h) and late (48 h) inflammatory responses caused by carrageenan (Cg) injected into the mouse pleural cavity. The i.pl. injection of IL-6, 5 min prior to Cg, reduced in a dose-dependent and significant manner, the exudation and total and differential leukocyte migration according to assessment in both the early (4 h) and the late (48 h) phases of Cg inflammatory response (P<0.01). Intrapleural injection of IL-10, 5 min prior to i.pl. injection of Cg, resulted in a significant inhibition of the early phase (4 h) (P<0.01), but had no significant effect in relation to the late (48 h) phase of Cg response. The antibodies anti-IL-6 (given i.pl. 30 min prior to Cg) caused a significant decrease in both total and differential leukocyte influx, but significantly increased exudation according to assessment 4 h after pleurisy induction by Cg (P<0.01). In contrast, anti-IL-10 antibody caused graded and marked increase of both total and differential leukocyte influx and also increased fluid leakage as assessed 4 h after Cg injection (P<0.01). In the late phase (48 h) these antibodies increased the inflammatory parameters (anti-IL-6) studied or had no effect (anti-IL-10). Taken together, the current results confirm and extend previous data from the literature by showing that IL-6 and IL-10 regulate several signs of inflammatory response, here characterized by marked inhibition of polymorphonuclear cell influx and blockage of fluid leakage to the site of Cg-induced pleurisy in the mouse.

Generation of cytotoxic T lymphocytes by MHC class I ligands fused to heat shock cognate protein 70.

Immunization with gp96 and heat shock cognate protein 70 (hsc70) purified with in vivo bound naturally occurring peptides or bound to synthetic peptides by in vitro reconstitution has been shown to induce peptide-specific cytotoxic T lymphocytes (CTL). In addition, mycobacterial heat shock protein 70 covalently fused to ovalbumin (OVA)-derived fragments has been shown to generate MHC class I-restricted CTL responses. Here, we genetically fused five different CTL epitopes, including peptides derived from Plasmodium yoelii circumsporozoite protein, tumor antigens, HY antigen and OVA, to either the N- or C-terminus of murine hsc70 and expressed the resulting proteins in Escherichia coli. Vaccination with all five fusion proteins induced peptide-specific CTL, indicating that no cognate flanking regions of CTL epitopes are necessary for the immune response. The point of injection was crucial for CTL induction. CD4(+) T cells were not required for the priming of CD8(+) T cells and vaccination with bone marrow-derived dendritic cells pulsed with hsc70 fusion proteins also elicited CTL responses. Furthermore, by using deletion mutants of hsc70, we identified amino acid residues 280-385 of hsc70 as the region most critical for inducing the CTL response.