Expression Of Cytokine-induced Neutrophil Chemoattractant Research Articles

Aging increases the expression of lung CINCs and MCP-1 in senile patients with pneumonia.

ObjectiveTo explore the relationship between aging and the expression of monocyte chemoattractant protein (MCP) and cytokine-induced neutrophil chemoattractant (CINCs) in patients with pneumonia.ResultsBacteria counts in senile group were significantly higher than non-senile group, and while white blood cell and neutrophil counts in senile group were observably lower than non-senile group. The concentration of MCP-1 was significantly higher in senile group compared with the non-senile group, and the expression of CINC-1 and CINC-2α was also higher in senile group. In all patients with different pathogens, expression of all the factors was significantly higher in senile group compared with the non-senile group. What’s more, expression of MCP-1, CINC-1 and CINC-2α showed significant difference in some patients with different pathogens. CINC-2β and CINC-3 expression was not detected in both groups.Materials and methodsThe present study included 800 patients with pneumonia who were hospitalized to the Department of Respiratory Medicine in Tongji Hospital during the period from December of 2014 to June of 2016. All patients were divided into two groups: senile pneumonia and non-senile pneumonia group. Bacteria, white blood cell and neutrophil counts were determined by automatic blood cell analyzer. The expression of MCP-1, CINC-1, CINC-2α, CINC-2β and CINC-3 was determined by ELISA assay.ConclusionsAging can increase the expression of MCP-1,CINC-1 and CINC-2α in patients with pneumonia, which may lead to increased risk of pneumonia in the elderly.

Regulatory effect of cytokine-induced neutrophil chemoattractant, epithelial neutrophil-activating peptide 78 and pyrrolidine dithiocarbamate on pulmonary neutrophil aggregation mediated by nuclear factor-κB in lipopolysaccharide-induced acute respiratory distress syndrome mice.

In the present study, the regulatory effect of cytokine-induced neutrophil chemoattractant (CINC) and epithelial neutrophil-activating peptide 78 (ENA-78) on pulmonary neutrophil (PMN) accumulation in lipopolysaccharide (LPS)-induced acute respiratory distress syndrome (ARDS) mice, and the therapeutic effect of pyrrolidine dithiocarbamate (PDTC), was investigated. BALB/c mice were divided into control, LPS and PDTC + LPS groups using a random number table. The phosphorylation of nuclear factor-κB (NF-κB) was detected using a western blot, and the mRNA expression levels of CINC were evaluated using reverse transcription-quantitative polymerase chain reaction. The expression of NF-κB, CINC and ENA-78 was detected using immunohistochemistry. The production of interleukin (IL)-8 and IL-10 in serum and broncho-alveolar lavage fluid (BALF) was analyzed using an enzyme-linked immunosorbent assay. The total number of leukocytes and proportion of PMNs in BALF was also determined. Following injection with LPS (20 mg/kg), the expression levels of p-NF-κB, CINC and ENA-78 were increased in lung tissue, and the expression levels of IL-8, IL-10 and the number of PMNs increased in serum and BALF. However, in comparison with the LPS group, the degree of lung injury was reduced in ARDS mice that were treated with PDTC. In addition, the expression level of p-NF-κB and the production of chemokines in lung tissue decreased in ARDS mice that were treated with PDTC, and the number of PMNs in BALF also decreased. In conclusion, the results of the present study suggest that the LPS-induced phosphorylation of NF-κB may result in the synthesis and release of CINC and ENA-78, which induce the accumulation of PMNs in the lung. Therefore, PDTC may be used to reduce the production of chemokines and cytokines, thereby decreasing the activation of PMNs in lung tissue and reducing the damage of lung tissue in ARDS.

Uterine cytokine profile in a rat model of endometritis.

Endometritis is a common reproductive disorder in female domestic animals. Roles of cytokines and chemokines have been implicated in this disease. To date, no comprehensive panel of the cytokine profile in inflammatory sites of endometritis has been reported. To address cytokine profiles in endometritis, a bacteria-induced rat model of endometritis was developed and levels of 27 cytokines were measured in paired uterine horn tissues using a multiple cytokine array. Of the 27 cytokines, five pro-inflammatory mediators, including three cytokine-induced neutrophil chemo-attractant (CINC)-1, CINC-2 and CINC-3, interleukin (IL)-1a and CXC family member CXCL5/LIX were increased upon the stimulation of bacteria. High expression of CINCs as well IL-1a and CXCL5/LIX suggests their potent roles in the pathogenicity of endometritis.

Aging Reduces the Expression of Lung CINC and MCP-1 mRNA in a P. aeruginosa Rat Model of Infection

We investigated dynamic changes of inflammatory cell infiltration and expression of cytokine-induced neutrophil chemoattractant (CINC) and monocyte chemoattractant protein-1 (MCP-1) mRNA in aged rats with Pseudomonas aeruginosa pulmonary infection. Disease manifestation and lung tissue pathology (lesion dispersion, inflammatory reactions, tissue edema and bleeding) were more severe in aged rats than young rats. At various time points, lung tissue polymorphonuclear neutrophil and mononuclear macrophage numbers were lower in the aged group than the young group (P < 0.05), and at 24 h there was no difference in mononuclear macrophage numbers. After inoculation with P. aeruginosa, CINC and MCP-1 mRNA expression increased in both groups, but the peak lagged in old rats compared with young. Thus, aging can reduce the expression of CINC and MCP-1 mRNA in lung tissues, and reduce the infiltration of neutrophils and monocyte–macrophages induced by CINC and MCP-1. This might lead to increased risk of pneumonia in elderly patients.

Expression of cytokine-induced neutrophil chemoattractant in rat lungs following an intratracheal instillation of micron-sized nickel oxide nanoparticle agglomerates

In our previous study, we reported that the micron-sized nickel oxide nanoparticle agglomerates induced neutrophil infiltration and the gene expression of the cytokine-induced neutrophil chemoattractant (CINC)-2αβ in a rat lung. In this study, we examined the expression of the CINCs family in the lung using the same rat model exposed to micron-sized nickel oxide nanoparticle agglomerates. The count median diameter of nickel oxide nanoparticle agglomerates suspended in saline was 1.34 μm (primary diameter: 8.41 nm). Male Wistar rats received an intratracheal instillation of 1 mg (3.3 mg/kg) of nickel oxide nanoparticles and were dissected at 3 days, 1 week, 1 month, 3 months, and 6 months after the instillation. The negative control group received an instillation of saline. The concentration of CINC-1 in the lung and the bronchoalveolar lavage fluid (BALF), CINC-2αβ in the BALF, and CINC-3 in the lung and the BALF was examined. The concentration of CINC-1 was elevated at 3 days, 3 months, and 6 months in the lung tissue and from 3 days to 6 months in the BALF. The concentration of CINC-2αβ was elevated from 3 days to 3 months in the BALF. The concentration of CINC-3 was also elevated at 3 days, 1 week, 3 months, and 6 months in the lung tissue. Infiltration of neutrophil and alveolar macrophage was observed mainly in the alveoli during the observed time period. These results suggest that CINC-1 to -3 were totally involved in the lung injury caused by micron-sized nickel oxide nanoparticle agglomerates.

Colonic insufflation with carbon monoxide gas inhibits the development of intestinal inflammation in rats

BackgroundThe pathogenesis of inflammatory bowel disease (IBD) is complex, and an effective therapeutic strategy has yet to be established. Recently, carbon monoxide (CO) has been reported to be capable of reducing inflammation by multiple mechanisms. In this study, we evaluated the role of colonic CO insufflation in acute colitis induced by trinitrobenzene sulfonic acid (TNBS) in rats.MethodsAcute colitis was induced with TNBS in male Wistar rats. Following TNBS administration, the animals were treated daily with 200 ppm of intrarectal CO gas. The distal colon was removed to evaluate various parameters of inflammation, including thiobarbituric acid (TBA)-reactive substances, tissue-associated myeloperoxidase (MPO) activity, and the expression of cytokine-induced neutrophil chemoattractant (CINC)-1 in colonic mucosa 7 days after TNBS administration.ResultsThe administration of TNBS induced ulceration with surrounding edematous swelling in the colon. In rats treated with CO gas, the colonic ulcer area was smaller than that of air-treated rats 7 days after TNBS administration. The wet colon weight was significantly increased in the TNBS-induced colitis group, which was markedly abrogated by colonic insufflation with CO gas. The increase of MPO activity, TBA-reactive substances, and CINC-1 expression in colonic mucosa were also significantly inhibited by colonic insufflation with CO gas.ConclusionsColonic insufflation with CO gas significantly ameliorated TNBS-induced colitis in rats. Clinical application of CO gas to improve colonic inflammatory conditions such as IBD might be useful.

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To observe the effect of sevoflurane pretreatment with different concentrations on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and expression of heme oxygenase-1 (HO-1). Sprague-Dawley (SD) rats were divided into six groups in different sevoflurane pretreatment experiment, Group A (control), Group B (1.5 MAC sevoflurane), Group C (1.5 MAC sevoflurane + LPS), Group D (1.0 MAC sevoflurane + LPS), Group E (0.5 MAC sevoflurane + LPS) and Group F (LPS) at random. Besides, SD rats were divided into other six groups in induced HO-1 expression experiment, Group A (control group), Group B (ZnPP), Group C (1.0 MAC sevoflurane), Group D (1.0 MAC sevoflurane + LPS), Group E (ZnPP + 1.0 MAC sevoflurane + LPS) and Group F (LPS). Using traditional method to detect pathological changes, wet/dry ratio (W/D), myeloperoxidase (MPO) and HO-1 activity; detecting the mRNA and protein level of cytokine-induced neutrophil chemoattractant (CINC), and intercellular adhesion molecule-1(ICAM-1) and HO-1 by&nbsp;RT-PCR and Western blot. 1.0 MAC sevoflurane pre-treatment could effectively protect ALI with decreasing pathomorphological scores, MPO activity, W/D and down-regulated expression of ICAM-1 and CINC. Furthermore, 1.0 MAC sevoflurane pre-treatment could significantly induced HO-1 expression suggesting sevoflurane may protect ALI from inflammation through inducing abundant HO-1 expression. Sevoflurane pre-treatment may be an effective avenue to alleviate ALI through inducing HO-1 expression. &nbsp; Key words:&nbsp;Sevoflurane, acute lung injury, heme oxygenase-1 (HO-1).

Expression of cytokine-induced neutrophil chemoattractant in rat lungs by intratracheal instillation of nickel oxide nanoparticles

Since nanoparticles easily agglomerate to form larger particles, it is important to maintain the size of their agglomerates at the nano-level to evaluate the harmful effect of the nanoparticles. We prevented agglomeration of nickel oxide nanoparticles by ultrasound diffusion and filtration, established an acute exposure model using animals, and examined inflammation and chemokine expression. The mass median diameter of nickel oxide nanoparticle agglomerates suspended in distilled water for intratracheal instillation was 26 nm (8.41 nm weighted average surface primary diameter). Male Wistar rats received intratracheal instillation of nickel oxide nanoparticles at 0.1 mg (0.33 mg/kg) or 0.2 mg (0.66 mg/kg), and were dissected 3 days, 1 week, 1 month, 3 months, and 6 months after the instillation. The control group received intratracheal instillation of distilled water. Three chemokines (cytokine-induced neutrophil chemoattractant-1 (CINC-1), CINC-2αβ, and CINC-3) in the lung tissue and bronchoalveolar lavage fluid (BALF) were determined by quantitative measurement of protein by ELISA. Both CINC-1 and CINC-2αβ concentration was elevated from day 3 to 3 months in lung tissue and from day 3 to 6 months in BALF. On the other hand, CINC-3 was elevated on day 3 in both lung tissue and BALF, and then decreased. The total cell and neutrophil counts in BALF were increased from day 3 to 3 months. In lung tissue, infiltration of mainly neutrophils and alveolar macrophages was observed from day 3 to 6 months in alveoli. These results suggest that CINC was involved in lung injury by nickel oxide nanoparticles.

Dai‐kenchu‐to attenuates rat sinusoidal obstruction syndrome by inhibiting the accumulation of neutrophils in the liver

Sinusoidal obstruction syndrome (SOS) is drug-induced liver injury that occurs in patients who receive hematopoietic cell transplantation and oxaliplatin-contained chemotherapy. The aim of study was to investigate the pharmacological treatment of SOS using a traditional Japanese medicine, Dai-kenchu-to (DKT). Male Sprague-Dawley rats were treated with monocrotaline (MCT) to induce SOS. The rats were divided into three groups: control, MCT and MCT+DKT groups. In the MCT+DKT group, DKT was gavaged at 12 h after MCT treatment and given every 12 h until the end of the protocol. The rats of MCT group were treated with water instead of DKT. At 48 h after MCT treatment, blood and liver samples were collected. In the MCT+DKT group, the macroscopic and histological findings revealed liver congestion, sinusoidal alteration and the destruction of sinusoidal lining, which were comparable with those of the MCT group. However, the area of hepatic necrosis and serum AST levels significantly decreased in the MCT+DKT group compared with those of the MCT group. Treatment with DKT resulted in the reduction of neutrophil accumulation, myeloperoxidase activity and the expression of cytokine-induced neutrophil chemoattractant (CINC) and intracellular adhesion molecule-1 (ICAM-1) mRNA in the liver compared with those of the MCT group. Treatment with processed ginger, one of the ingredients in DKT, resulted in similar effects to those shown by DKT. Dai-kenchu-to attenuates MCT-induced liver injury by preventing neutrophil-induced liver injury through blockage of upregulation of CINC and ICAM-1 mRNA level.

Therapeutic effects of emodin on acute cholestatic hepatitis and mechanism thereof: experiment with rats

To investigate the therapeutic effects of emodin on acute cholestatic hepatitis and mechanism thereof. 96 SD rats were randomly divided into 4 groups to be treated with emodin, ursodeoxycholic acid, dexamethasone, or normal saline respectively for 4 days. On the 5th day gastric perfusion of alpha-naphthylisothiocyanate (ANIT) was performed to establish models of cholestatic hepatitis. 4 - 6 hours after the establishment of model the above mentioned agents were given continuously. 24, 48, and 72 hours after the model establishment blood samples were collected from abdominal aorta to examine the total bilirubin (TB), direct bilirubin (DB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyltransferase (GGT), and total bile acid (TBA). Specimen of liver was collected to undergo pathological examination. PCR was used to detect the mRNA expression of cytokine-induced neutrophil chemoattractant 1 (CINC-1) and macrophage inflammatory protein 2(MIP-2), and Western blotting was used to detect the protein expression of intercellular adhesion molecule 1 (ICAM-1). Twenty-four rats treated with NS only were used as controls. The pathological changes of behaviors and liver of the emodin and ursodeoxycholic acid groups were all remarkably milder than those of other model groups. The levels of TB [(32.8 +/- 3.7) micromol/L, (61.0 +/- 16.4) micromol/L, (10.8 +/- 4.5) micromol/L], DB[ (26.03 +/- 3.10) micromol/L, (49.40 +/- 18.16) micromol/L, (8.04 +/- 3.03) micromol/L], and ALT [(314 +/- 50) U/L, (664 +/- 97) U/L, (200 +/- 60) U/L], at the time points 24, 48, and 72 hours, the 48 and 72 hours AST levels, the 48 hours ALP level, the 72 hours GGT level, and the 48 and 72 hours TBA levels of the emodin group were all significantly lower than those of the model group (all P < 0.05). The 24 and 48 hours TB levels, 24 hours DB and ALT, and 24, 72 hours TBA levels of the emodin group were all significantly lower than those of the ursodeoxycholic acid group (all P < 0.05). The 24, 48 hours TB and TBA levels, and the ALT, AST and GGT levels at all time points of the emodin group were all significantly lower than those of the dexamethasone group (all P < 0.05). The CINC-1 and MIP-2 mRNA expression levels and ICAM-1 protein expression levels at all time points of the emodin group were all significantly lower than those of the model group (all P < 0.05). Decreasing the levels of TB, DB and ALT in particular, emodin has a protective effect on cholestatic hepatitis. Its effects are quicker than ursodeoxycholic acid, and it has better effects on ALT, AST GGT, and TBA than dexamethasone. These effects may be due to inhibition of the activation of CINC-1, MIP-2, and ICAM-1.

Mechanisms Involved in the Enhancement of Allergic Airways Neutrophil Influx by Permanent C-Fiber Degeneration in Rats

This study was undertaken to clarify the mechanisms by which C-fiber degeneration at neonatal stages exacerbates the inflammatory responses of rat airways. Rats were treated with capsaicin at neonatal stages and immunized with ovalbumin (OVA) at adult ages. Challenge of capsaicin-pretreated rats with OVA promoted a higher influx of neutrophils in bronchoalveolar lavage (BAL) fluid compared with the vehicle group. No significant differences were found for the other cell types. The increased adhesion of N-formyl-methionyl-leucyl-phenylalanine (fMLP; 0.1 microM)- and phorbol myristate acetate (PMA; 1 microM)-treated neutrophils to fibronectin-coated wells did not differ among vehicle- and capsaicin-pretreated rats. Additionally, fMLP (10 microM), platelet-activating factor (0.1 microM), and substance P (50 microM) induced a significant neutrophil chemotaxis, but no differences were found among vehicle and capsaicin groups. Increased levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-10, and leukotriene B4 in BAL fluid as well as higher expression of cytokine-induced neutrophil chemoattractant (CINC)-3 in lung homogenates were detected in the capsaicin group compared with vehicle group. In the capsaicin group, chronic treatment with compound 48/80 restored the TNF-alpha levels to control values and prevented the neutrophil influx in BAL fluid. The enhanced production of TNF-alpha, superoxide anion, and nitrite by isolated alveolar macrophages in response to lipopolysaccharide (3 microg/ml), PMA (10 nM), and/or zymosan (100 particles/cell) did not differ between vehicle- and capsaicin-pretreated rats. In conclusion, chronic neuropeptide depletion promoted by neonatal capsaicin treatment up-regulates airways mast cells, which upon activation by antigen at adult ages, release large amounts of cytokines such as TNF-alpha and CINC-3 that accounts for the massive airways neutrophil infiltration.

Impaired induction of IL-10 expression in the lung following hemorrhagic shock.

The balance between pro- and anti-inflammatory cytokines is considered to be an important determinant of the magnitude of inflammation in a number of disease states. We previously showed that resuscitated hemorrhagic shock augmented LPS-induced release of proinflammatory molecules by alveolar macrophages (AM). In the present studies, we evaluated the expression and regulation of the counter inflammatory cytokine IL-10 in the lung using this model. We hypothesized that impaired up-regulation of IL-10 in shock/resuscitated animals might serve as a mechanism contributing to accentuated lung inflammation. In a rodent model, animals exposed to LPS alone exhibited enhanced IL-10 mRNA levels in lung tissue as well as in AM, but antecedent shock/resuscitation delayed and attenuated the LPS-induced IL-10 mRNA levels. The ability of shock to attenuate LPS-stimulated IL-10 was also seen in the protein levels. This effect correlated with an augmented expression of cytokine-induced neutrophil chemoattractant (CINC) mRNA. Shock/resuscitated animals given exogenous IL-10 had reduced proinflammatory response, as shown by decreased expression of CINC mRNA and decreased neutrophil sequestration in the lung. Shock/resuscitation plus LPS markedly reduced the transcription rate of IL-10 mRNA compared to LPS alone but did not affect IL-10 mRNA stability. Reduced IL-10 transcription was not caused solely by impaired nuclear translocation of STAT3 and Sp1/Sp3 transcription factors because LPS-induced nuclear translocation of these factors was augmented by antecedent shock. Considered together, these findings show that shock/resuscitation suppresses LPS-induced IL-10 expression by AM in the lung by inhibiting IL-10 gene transcription. Failed up-regulation of counter inflammatory cytokines may contribute to augmented organ dysfunction in trauma patients.

Alpha chemokines regulate direct lung ischemia–reperfusion injury

Background Alpha chemokines function predominantly to recruit and activate neutrophils, which are important effectors of acute lung injury. This study evaluated whether blockade of 2 potent alpha chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC), is protective against lung ischemia–reperfusion injury in a warm in situ hilar clamp model. Methods Left lungs of Long–Evans rats underwent normothermic ischemia for 90 minutes and reperfusion for up to 4 hours. Treated animals received antibodies to MIP-2 or CINC immediately prior to reperfusion. Lung injury was quantitated by vascular permeability to 125I-radiolabeled bovine serum albumin, lung tissue neutrophil sequestration (myeloperoxidase [MPO] content), and alveolar leukocyte content in bronchoalveolar lavage (BAL) fluid. CINC and MIP-2 mRNA expression were assessed by northern blot, while ribonuclease protection assays were performed to evaluate mRNA expression for a number of early response cytokines. MIP-2 and CINC protein expression in injured lungs was determined by immunoblotting. Results Treatment with antibodies to CINC or MIP-2 was associated with significant protection against increases in vascular permeability, MPO content and alveolar leukocyte sequestration in injured lungs. Expression of CINC and MIP-2 mRNA peaked after 2 hours of reperfusion in injured lungs, and protein levels were evident on immunoblotting after 3 hours of reperfusion. Neither CINC nor MIP-2 blockade appeared to modulate cytokine mRNA expression. Conclusions CINC and MIP-2 are important mediators involved in direct lung ischemia–reperfusion injury. They appear to function by modulating neutrophil recruitment, but not inflammatory cytokine release.

Protective Effects of Urinary Trypsin Inhibitor (UTI) on Hepatic Microvasculature in Hypotensive Brain-Dead Rats

Background/Aims: We examined the protective effects of a protease inhibitor, urinary trypsin inhibitor (UTI), on livers of brain dead rats associated with systemic hemodynamic instability. Methods: Brain death was induced by inflating a balloon catheter placed in the epidural space in rats followed by intravenous administration of UTI for 6 h. The hemodynamic, functional and morphological changes in the liver were examined. Results: The induction of brain death resulted in a significant decrease in both mean arterial pressure (MAP) and hepatic tissue flow (HTF), and an increase in serum AST and cytokines such as tumor necrosis factor (TNF)-α and cytokine-induced neutrophil chemoattractant (CINC). An increase in the number of sequestered neutrophils and enhanced expressions of intercellular adhesion molecules (ICAM)-1 and CINC were also noted in the liver. The treatment with UTI significantly restored HTF to basal level without affecting MAP, and decreased the number of sequestered neutrophils in the hepatic sinusoids, suppressed the expression of ICAM-1 and CINC in the sinusoids, inhibited the production of serum TNF-α and CINC, and inactivated Kupffer cells. Conclusion: Intravenous administration of UTI is likely to ease unfavorable effects on the hepatic microvasculature evoked by brain death.

Effect of cold-ischemia time on C-X-C chemokine expression and neutrophil accumulation in the graft liver after orthotopic liver transplantation in rats

The precise mechanisms leading to polymorphonuclear neutrophil (PMN) recruitment and activation in the extended cold-preserved liver after transplantation are not yet fully understood. We histologically evaluated the number of accumulated PMNs in graft livers, with varying time periods of cold ischemia (1, 6, and 24 hr in University of Wisconsin solution at 4 degrees C), after liver transplantation in rats. Intragraft expression of macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) mRNA, as well as immunohistochemical expression of MIP-2 and CINC in the graft liver, were investigated after reperfusion. The levels of MIP-2 and CINC in the hepatic vein, and tumor necrosis factor (TNF)-alpha, which stimulates these chemokine production, were also monitored. The number of accumulated PMNs in sinusoids significantly increased in the 24-hr cold-ischemia group within 3 hr after reperfusion, compared with the 1-hr and 6-hr groups. Serum MIP-2 levels in the 24-hr group significantly increased at 3, 6, and 12 hr after reperfusion, compared with the other groups. Intragraft MIP-2 mRNA was also up-regulated to a greater extent in the 24-hr group. Similarly, serum CINC levels in the 24-hr group significantly increased at 3 hr, compared with the 1-hr group. CINC mRNA also increased as cold-ischemia time was prolonged. Immunohistochemical staining revealed that hepatocytes were the main source of both MIP-2 and CINC protein. In addition, TNF-alpha in the hepatic vein was detected only in the 24-hr group after reperfusion. Extended cold preservation of the graft liver might up-regulate MIP-2 and CINC expression of hepatocytes, most probably through elevated TNF-alpha, and might contribute to PMN recruitment and activation after reperfusion.

Sublethal Endotoxemia Promotes Pulmonary Cytokine-Induced Neutrophil Chemoattractant Expression and Neutrophil Recruitment but Not Overt Lung Injury in Neonatal Rats

Gram-negative sepsis and septic shock remain major causes of morbidity and mortality in the newborn. Respiratory failure is a common feature in neonatal sepsis regardless of the presence or absence of associated pneumonia. In adult animal models, cytokine-induced neutrophil chemoattractant (CINC) is a potent chemoattractant for neutrophils and believed to play a role in endotoxin-induced lung injury. We examined this in a neonatal model. Ten-day-old Sprague-Dawley rats were injected with Salmonella enteritidis endotoxin (ETX) 0.03 mg/kg i.p. and sacrificed at baseline, 30 min, 1, 2, 4, 8 and 16 h post-ETX. Blood was collected by cardiac puncture. After bronchoalveolar lavage, lung tissue was collected and evaluated for neutrophil (polymorphonuclear leukocyte) recruitment by myeloperoxidase assay (MPO). Lung CINC expression was measured by Northern blot and ELISA. Peripheral blood leukocytosis was noted at 1 h (p < 0.001) with counts below baseline at 2 and 4 h. Differential counts revealed neutrophilia at 8 h (p < 0.001). MPO revealed pulmonary PMN recruitment peaking at 1 h (p < 0.05) and CINC RNA and protein expression peaked slightly later at 2 h (p < 0.001). No overt lung injury was noted by bronchoalveolar lavage cell counts or by histology. Therefore, pulmonary CINC expression and neutrophil recruitment follows LPS exposure in neonatal rats. This may represent priming of the lung tissue and a secondary event may be necessary for injury to occur

Thrombin and factor Xa enhance neutrophil chemoattractant production after ischemia/reperfusion in the rat liver.

Clotting proteases may affect leukocyte effector function. Activation of the coagulation cascade after ischemia/reperfusion stimulates cytokine production by activated macrophages. Cytokine-induced neutrophil chemoattractant (CINC) may also be important in the pathophysiology of liver ischemia/reperfusion injury. We investigated the effects of a selective factor Xa inhibitor, DX-9065a, on CINC expression after ischemia/reperfusion in the rat liver. Liver ischemia was induced in rats by occluding the portal vein for 30 min. DX-9065a (9 mg/kg) was injected intravenously 5 min before vascular clamping. Serum CINC concentrations were measured by enzyme-linked immunosorbent assay. Levels of CINC mRNA in the liver were determined by Northern blot analysis. We also examined in vitro CINC production by peritoneal macrophages in response to alpha-thrombin or factor Xa. Serum CINC concentrations increased and peaked 6 h after reperfusion. However, pretreatment of animals with DX-9065a resulted in significantly smaller increases in CINC after reperfusion. Pretreatment with DX-9065a also significantly reduced CINC mRNA levels in the liver after ischemia/reperfusion. In vitro CINC production by peritoneal macrophages was enhanced by alpha-thrombin, as well as factor Xa. Thrombin and factor Xa stimulate CINC production by macrophages. A selective inhibitor of factor Xa, DX-9065a, attenuates neutrophil chemoattractant production after ischemia/reperfusion injury of the rat liver.

Reduction of oxidative stress and cytokine-induced neutrophil chemoattractant (CINC) expression by red wine polyphenols in zinc deficiency induced intestinal damage of rat

Consumption of red wine has been associated with health promotion and disease prevention. We have previously found that the intestine of zinc-deficient (ZD) rats develop oxidative damage associated with inflammation. Here we have used this model to investigate whether red wine polyphenols could protect against intestinal injury and, if so, whether this protection was achieved through antioxidant and anti-inflammatory activity. The intestinal alterations induced by zinc deficiency such as morphological damage, increased TBA-RS level and CuZn-superoxide dismutase activity, and decreased glutathione peroxidase activity, did not develop with the administration to ZD rats of a suspension of dealcoholated red wine (RWS). The same treatment induced in control rats a decrease of TBA-RS level but also of glutathione peroxidase and catalase activity. Treatment with RWS to ZD rats prevented a marked mucosal macrophage and neutrophil infiltration. The expression of pro-inflammatory cytokines, such as tumor necrosis factor α and cytokine-induced neutrophil chemoattractant (CINC), was induced by zinc deficiency, whereas that of the anti-inflammatory interleukin-10 was suppressed. Treatment with RWS reduced CINC expression. These results report a novel activity of red wine polyphenols in downregulation of intestinal CINC expression, which likely protects cells against inflammatory processes.

Bile duct ligation in rats induces biliary expression of cytokine-induced neutrophil chemoattractant

Background & Aims: Bile duct obstruction causes neutrophilic inflammation of the liver and leads to hepatic fibrosis. In obstructive liver disease, the localization of neutrophils in portal tracts suggests that cells within this region produce neutrophil chemoattractants. In this study, we investigated whether bile duct obstruction in rats induces portal expression of cytokine-induced neutrophil chemoattractant (CINC). Methods: Rats underwent bile duct ligation for 3 hours to 8 days. CINC regulation was examined in vivo at various intervals by immunohistochemistry, ribonuclease protection, and in situ hybridization. CINC production was also investigated in cell culture, in response to putative stimuli from obstructed liver. Results: Bile duct ligation caused neutrophilic infiltration of the liver within 3 hours. CINC was also rapidly induced, with specific expression identified in biliary cells. Rat intrahepatic biliary cells produced CINC constitutively in culture; when exposed to cholestatic bile, they showed a 12-fold increase in CINC secretion. The effect of bile was not attributable to toxicity or to dissolved cytokines or endotoxin. Mechanical strain, designed to mimic the stretching of biliary cells during obstruction, did not induce CINC. Conclusions: Biliary cells contribute to hepatic inflammation during cholestasis by producing neutrophil chemoattractants. A major stimulus to biliary chemoattractant production in vivo may be bile itself. GASTROENTEROLOGY 2000;118:1157-1168

Transient Nuclear Factor κB (NF-κB) Activation Stimulated by Interleukin-1β May Be Partly Dependent on Proteasome Activity, but Not Phosphorylation and Ubiquitination of the IκBα Molecule, in C6 Glioma Cells: REGULATION OF NF-κB LINKED TO CHEMOKINE PRODUCTION

We previously reported that several stresses can induce cytokine-induced neutrophil chemoattractant expression in a nuclear factor kappaB (NF-kappaB)-dependent manner. In this study, we focused further on the regulation of NF-kappaB. The activation of NF-kappaB and the subsequent cytokine-induced neutrophil chemoattractant induction in response to interleukin-1beta (IL-1beta) were inhibited by proteasome inhibitors, MG132 and proteasome inhibitor I. Translocation of NF-kappaB into nuclei occurs by the phosphorylation, multi-ubiquitination, and degradation of IkappaBalpha, a regulatory protein of NF-kappaB. Nascent IkappaBalpha began to degrade 5 min after treatment with IL-1beta and disappeared completely after 15 min. However, IkappaBalpha returned to basal levels after 45-60 min. Interestingly, resynthesized IkappaBalpha was already phosphorylated at Ser-32. These results suggest that 1) the upstream signals are still activated, although the translocation of NF-kappaB peaks at 15 min; and 2) the regulated protein(s) acts downstream of IkappaBalpha phosphorylation. Western blotting showed that the resynthesized and phosphorylated IkappaB molecules were also upward-shifted by multi-ubiquitination in response to IL-1beta treatment. On the other hand, ATP-dependent Leu-Leu-Val-Tyr cleaving activity transiently increased, peaked at 15 min, and then decreased to basal levels at 60 min. Furthermore, the cytosolic fraction that was stimulated by IL-1beta for 15 min, but not for 0 and 60 min, could degrade phosphorylated and multi-ubiquitinated IkappaBalpha. These results indicate that the transient translocation of NF-kappaB in response to IL-1beta may be partly dependent on transient proteasome activation.