Characterization of the MEK5-ERK5 module in human neutrophils and its relationship to ERK1/ERK2 in the chemotactic response

The role of the extracellular signal-regulated kinase (ERK) 1 and ERK2 in the neutrophil chemotactic response remains to be identified since a previously used specific inhibitor of MEK1 and MEK2, PD98059, that was used to provide evidence for a role of ERK1 and ERK2 in regulating chemotaxis, has recently been reported to also inhibit MEK5. This issue is made more critical by our present finding that human neutrophils express mitogen-activated protein (MAP) kinase/ERK kinase (MEK)5 and ERK5 (Big MAP kinase), and that their activities were stimulated by the bacterial tripeptide, formyl methionyl-leucyl-phenylalanine (fMLP). Dose response studies demonstrated a bell-shaped profile of fMLP-stimulated MEK5 and ERK5 activation, but this was left-shifted when compared with the profile of fMLP-stimulated chemotaxis. Kinetics studies demonstrated increases in kinase activity within 2 min, peaking at 3–5 min, and MEK5 activation was more persistent than that of ERK5. There were some similarities as well as differences in the pattern of activation between fMLP-stimulated ERK1 and ERK2, and MEK5-ERK5 activation. The up-regulation of MEK5-ERK5 activities was dependent on phosphatidylinositol 3-kinase. Studies with the recently described specific MEK inhibitor, PD184352, at concentrations that inhibited ERK1 and ERK2 but not ERK5 activity demonstrate that the ERK1 and ERK2 modules were involved in regulating fMLP-stimulated chemotaxis and chemokinesis. Our data suggest that the MEK5-ERK5 module is likely to regulate neutrophil responses at very low chemoattractant concentrations whereas at higher concentrations, a shift to the ERK1/ERK2 and p38 modules is apparent.

Effects of dietary conjugated linoleic acid on haematological and humoral responses in the grower pig

The effects of conjugated linoleic acid (CLA) on the levels of total serum leucocytes, granulocytes including neutrophils, basophils, and eosinophils, as well as on monocytes and leucocytes were measured in pigs selected from a clean (minimal disease) herd. Thirty pigs were fed different rates of dietary CLA (0, 1.25, 2.5, 5.0, 7.5, and 10.0 g CLA-55/kg diet) for 8 weeks. Blood samples were collected at the end of the study for assessment of haematological and humoral responses to CLA supplementation. No difference in total white blood cells including the neutrophil, monocyte, and lymphocyte counts was observed among different dietary groups. A dose-dependent reduction (P = 0.02) in eosinophil concentrations suggests that CLA exerts anti-inflammatory activities. A 2-fold increase in the level of basophils was recorded in pigs fed lower levels of CLA (1.25 and 2.5 g CLA/kg diet) but the levels decreased gradually (P = 0.05) and were below the detection limit at the highest rate (10 g/kg) of CLA supplementation. The level of IgG was reduced by over 50% in CLA-fed pigs (P < 0.001), although the response was quadratic in nature (P < 0.001). T-cell population analysis showed that CD4+ cells tended (P = 0.06) to be reduced linearly with increasing inclusion of CLA in the diet. Our results suggest that dietary CLA modulates haematological and humoral responses in a dose-dependent manner.

Effect of Glucocorticoid pretreatment on oxidative liver injury and survival in jaundiced rats with Endotoxing Cholangitis

Introduction: Biliary tract infection is associated with high mortality. This study investigated the effect of glucocorticoid pretreatment on lipopolysaccharide (LPS)-induced cholangitis. Methods: Rats undergoing either sham operation or ligation of the extrahepatic bile duct (BDL) for 2 weeks were randomly assigned to receive intravenous injections of dexamethasone (DX) or normal saline (NS) prior to infusing LPS into the biliary tract. The plasma levels of tumor necrosis factor-α (TNFα), chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) as well as liver mRNA expression of MCP-1 and MIP-2 were determined. Infiltration of monocytes, Kupffer cells, and neutrophils in rat liver were studied with immunohistochemistry. Oxidative liver injury was measured by the malondialdehyde (MDA) content. Results: Dexamethasone pretreatment resulted in significantly decreased plasma levels of TNFα at 1 hour, MCP-1 and MIP-2 at 2 and 3 hours, and decreased liver MCP-1 mRNA expression at 3 hours following LPS infusion in BDL-DX rats than in BDL-NS rats. The number of inflammatory cells in the liver was significantly different between sham- and BDL-treated rats but was not affected by DX pretreatment. Pretreatment with DX resulted in significantly decreased liver MDA contents in the BDL-DX group than that in the BDL-NS group. Jaundiced rats pretreated with 5 mg DX prior to infusion of 1 g of LPS were 6.8 times more likely to survive than those that were not pretreated. Conclusions: Pretreatment of jaundiced, LPS-treated rats with a  supraphysiological dose of dexamethasone may rescue their lives by suppression of chemokine expression and alleviation of oxidative liver injury.

Biliary intervention augments chemotactic reaction and aggravates cholestatic liver injury in rats

Background
Intervention of the biliary system is frequently done in patients with obstructive jaundice and is associated with significant morbidity and mortality. The pathogenesis is unknown.
Materials and methods
A rat model of bile duct ligation (BDL) for 2 weeks was established in which biliary intervention was feasible by injection of normal saline through an indwelling catheter in the bile ducts. Plasma levels of C-C chemokine MCP-1 and C-X-C chemokine MIP-2 were measured by using ELISA. Blood monocytes, Kupffer cells, and neutrophils in the liver were characterized with antibodies to ED1, ED2, and myeloperoxidase (MPO). Lipid peroxidation was measured by malondialdehyde contents and apoptosis by TUNEL stain of the liver.
Results
Biliary intervention resulted in an increase of plasma MCP-1 and MIP-2 proteins by 1 h, which declined to normal level by 3 h in both sham and BDL rats. The levels in BDL rats were significantly higher than in sham at most points. There was a transient increase of ED1- and ED2-positive cells and MPO-staining cells in sham rat liver by 1 h after intervention. ED2-positive cells increased significantly by 1 h, while ED1- and MPO-positive cells decreased, yet insignificantly after intervention in BDL rats. The cell counts in BDL were constantly higher than in sham. Malondialdehyde increased precipitously in BDL by 3 h and was significantly higher than in sham throughout the study period. Parenchymal liver injury, manifested by elevated ALT, as well as apoptosis and necrosis of liver cells, was significantly increased in BDL rats, but not in sham rats.
Conclusion
Biliary intervention augments chemokine expression, precipitates lipid peroxidation, and aggravates liver injury in cholestatic rats.

Studies of the human lymphocyte P2Z receptor and its activation of phospholipase D

Extracellular adenosine 5′-triphosphate (ATP) is an agonist for the P2Z receptor of human leukaemic lymphocytes and opens a Ca ²⁺-selective ion channel, which also conducts Ba²⁺, Sr²⁺ and the small fluorescent dye, ethidium⁺. A wide range of receptor agonists, many of which raise cytosolic [Ca²⁺] activate phospholipase D (PLD). In the present study, it was shown that both ATP and 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) stimulated PLD activity in a concentration-dependent manner, and the inhibitory effects of suramin, oxidised ATP, extracellular Na⁺ and Mg²⁺ suggested that the effect of these agonists is mediated by P2Z receptors. The role of divalent cations in ATP-stimulated PLD activity was investigated. Several agonists (eg ATP, thapsigargin, ionomycin) stimulated a rise in cytosolic [Ca²⁺] in human lymphocytes, but only ATP and ionomycin stimulated PLD activity. When Ca²⁺ influx was prevented by EGTA, the majority of ATP-stimulated and all of ionomycin-stimulated PLD activity was inhibited. Preloading cells with the Ca²⁺ chelator, BAPTA, reduced cytosolic [Ca²⁺] and, paradoxically, ATP-stimulated PLD activity was potentiated. ATP-stimulated PLD activity was supported by both Ba²⁺ and Sr²⁺ when they were substituted for extracellular Ca²⁺. Furthermore, both ATP-stimulated PLD activity and ATP-stimulated ¹³³Ba²⁺ influx showed a linear dependence on extracellular [Ba²⁺]. Thus it was concluded that ATP stimulated PLD activity in direct proportion to the influx of divalent cations through the P2Z ion channel and this PLD activity was insensitive to changes in bulk cytosolic [Ca²⁺]. The calmodulin (Ca²⁺/CaM) inhibitor, trifluoperazine (TFP) inhibited ionomycin- and ATP-stimulated PLD activity and ATP-stimulated apoptosis, but had no effect on PLD activity already activated by ATP. However, TFP inhibited ATP-stimulated Ca²⁺, Ba²⁺ and ethidium⁺ fluxes, at concentrations below those which inhibit Ca²⁺/CaM, suggesting that TFP inhibits the P2Z receptor. Similarly, the isoquinolinesulphonamide, KN-62, a selective inhibitor of Ca²⁺/CaM-dependent protein kinase II (CaMKII), also prevented ATP-stimulated apoptosis, but had no effect on pre-activated PLD. In addition, KN-62, and an analogue, KN-04, which has no effect on CaMKII, potently inhibited ATP-stimulated Ba²⁺ influx (IC₅₀ 12.7 ± 1.5 and 17.3 ± 2.7 nM, respectively), ATP-stimulated ethidium⁺ uptake (IC₅₀ 13.1 ± 2.6 and 37.2 ± 8.9 nM, respectively), ATP-stimulated phospholipase D activity (50% inhibition 5.9 ± 1.2 and 9.7 ± 2.8 nM, respectively) and ATP-induced shedding of the surface adhesion molecule, L-selectin (IC₅₀ 31.5 ± 4.5 and 78.7 ± 10.8 nM, respectively). They did not inhibit phorbol ester- or ionomycin-stimulated PLD activity or phorbol ester-induced L-selectin shedding. Neither KN-62 nor KN-04 (both 500 nM) have any effect on UTP-stimulated Ca²⁺ transients in fura-2-loaded human neutrophils, a response which is mediated by the P2Y₂ receptor, neither did they inhibit ATP-stimulated contractile responses mediated by the P2X₁ receptor of guinea pig urinary bladder. Thus, KN-62 and KN-04 are almost equipotent as P2Z inhibitors with IC₅₀s in the nanomolar, indicating that their actions cannot be due to CaMKII inhibition, but rather that they are potent and direct inhibitors of the P2Z receptor. Extracellular ATP-induced shedding of L-selectin from lymphocytes into the medium is a Ca²⁺-independent response. L-selectin is either cleaved by a metalloproteinase or a PLD with specificity for glycosylphosphatidylinositol (GPI). The novel hydroxamic acid-based zinc chelator, Ro-31-9790 blocks ATP-induced L-selectin shedding, but was without effect on ATP-induced Ba²⁺ influx or ATP-stimulated PLD activity. Furthermore, another zinc chelator, 1,10-phenanthroline, an inhibitor of a GPI-PLD, potentiated rather than inhibited ATP-stimulated PLD activity, suggesting that ATP-induced L-selectin shedding and ATP-stimulated PLD activity are independent of each other. Although extracellular ATP is the natural ligand for the lymphocyte P2Z receptor, it is less potent than BzATP in stimulating Ba²⁺ influx. Concentration-response curves for BzATP- and ATP-stimulated ethidium⁺ influx gave EC₅₀s 15.4 ± 1.4 µM and 85.6 ± 8.8 µM, respectively. The maximal response to ATP was only 69.8 ± 1.9% of that for BzATP. Hill coefficients were 3.17 ± 0.24 and 2.09 ± 0.45 for BzATP and ATP respectively, suggesting greater positive cooperativity for BzATP than for ATP in opening the P2Z-operated ion channel. A rank order of agonist potency of BzATP > ATP = 2MeSATP > ATPγS was observed for agonist-stimulated ethidium⁺ influx, while maximal influxes followed a rank order of BzATP > ATP > 2MeSATP > ATPγS. When ATP (300 -1000 µM) was added simultaneously with 30 µM BzATP (EC₉₀), it reduced both ethidium⁺ and Ba²⁺ fluxes by 30 - 40% relative to values observed with BzATP alone. KN-62, previously shown to be a specific inhibitor of the lymphocyte P2Z receptor, was a less potent antagonist of BzATP-induced fluxes than ATP, when maximal concentrations of both agonists (50 and 500 µM respectively) were used. However, when BzATP (18 µM) was used at a concentration equiactive with a maximally effective ATP concentration, KN-62 showed the same inhibitory potency for both agonists. The ecto-ATPase antagonist, ARL-67156, inhibited both ATP- and BzATP-stimulated Ba²⁺ influx, suggesting that the lower efficacy of ATP compared with BzATP was not due to preferential hydrolysis of ATP. Thus, the natural ligand, ATP, is a partial agonist for the P2Z receptor while BzATP is a full agonist. Moreover the competitive studies show that only a single class of P2-receptor (P2Z class) is expressed on human leukaemic lymphocytes. Both ATP- and BzATP-stimulated PLD activity were significantly inhibited (P < 0.05) when cells were suspended in iso-osmotic choline Cl medium. Choline⁺ was found to be a permeant for the P2Z ion channel, since ATP induced a large uptake of [¹⁴C]choline⁺ (60 to 150 µmol/ml intracellular water) during a 5 min incubation, which remained in the cells for several hours, and ATP was used to load cells with these levels of choline⁺. Intracellular choline⁺ inhibited ATP-, BzATP-, PMA- and ionomycin-stimulated PLD activity. Brief exposure of lymphocytes to ATP increased the subsequent basal rate of ethidium⁺ uptake, and this was prevented by intracellular choline⁺. It is proposed that P2Z-mediated Ca²⁺ influx in lymphocytes activates PLD leading to significantly changes of the phospholipid composition of the plasma membrane, which subsequently produces a permeability lesion, which in turn contributes to cell death.

Bone marrow chimeras and c-fms conditional ablation (mafia) mice reveal an essential role for resident myeloid cells in lipopolysaccharide/TLR4-induced corneal inflammation

The mammalian cornea contains an extensive network of resident macrophages and dendritic cells. To determine the role of these cells in LPS-induced corneal inflammation, TLR4−/− mice were sublethally irradiated and reconstituted with bone marrow cells from either enhanced GFP (eGFP)+/C57BL/6 or eGFP+/TLR4−/− mice. The corneal epithelium was abraded, LPS was added topically, and cellular infiltration to the corneal stroma and development of corneal haze were examined after 24 h. TLR4−/− mice reconstituted with C57BL/6, but not TLR4−/− bone marrow cells donor cells were found to cause infiltration of eGFP+ cells to the cornea, including neutrophils, and also increased corneal haze compared with saline-treated corneas. In a second experimental approach, corneas of transgenic macrophage Fas induced apoptosis (Mafia) mice were stimulated with LPS. These mice express eGFP and a suicide gene under control of the c-fms promoter, and systemic treatment with the FK506 dimerizer (AP20187) causes Fas-mediated apoptosis of monocytic cells. AP20187-treated mice had significantly fewer eGFP+ cells in the cornea than untreated mice. After stimulation with LPS neutrophil recruitment and development of corneal haze were impaired in AP20187-treated mice compared with untreated controls. Furthermore, LPS induced CXCL1/KC and IL-1α production within 4 h in corneas of untreated Mafia mice, which is before cellular infiltration; however, cytokine production was impaired after AP20187 treatment. Together, results from both experimental approaches demonstrate an essential role for resident corneal monocytic lineage cells (macrophages and dendritic cells) in development of corneal inflammation.

Host defense at the ocular surface

Microbial infections of the cornea frequently cause painful, blinding and debilitating disease that is often difficult to treat and may require corneal transplantation. In addition, sterile corneal infiltrates that are associated with contact lens wear cause pain, visual impairment and photophobia. In this article, we review the role of Toll-Like Receptors (TLR) in bacterial keratitis and sterile corneal infiltrates, and describe the role of MD-2 regulation in LPS responsiveness by corneal epithelial cells. We conclude that both live bacteria and bacterial products activate Toll-Like Receptors in the cornea, which leads to chemokine production and neutrophil recruitment to the corneal stroma. While neutrophils are essential for bacterial killing, they also cause tissue damage that results in loss of corneal clarity. These disparate outcomes, therefore, represent a spectrum of disease severity based on this pathway, and further indicate that targeting the TLR pathway is a feasible approach to treating inflammation caused by live bacteria and microbial products. Further, as the P. aeruginosa type III secretion system (T3SS) also plays a critical role in disease pathogenesis by inducing neutrophil apoptosis and facilitating bacterial growth in the cornea, T3SS exotoxins are additional targets for therapy for P. aeruginosa keratitis.

Leukocyte numbers and function in subjects eating n-3 enriched foods: selective depression of natural killer cell levels

Introduction : While consumption of omega-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) has been recommended for those at risk of inflammatory disease such as rheumatoid arthritis, the mechanism of their anti-inflammatory effect remains to be clearly defined, particularly in relation to the dose and type of n-3 LCPUFA. The objective of this study was to determine whether varying the levels of n-3 LCPUFA in erythrocyte membrane lipids, following dietary supplementation, is associated with altered numbers and function of circulating leukocytes conducive to protection against inflammation. Methods : In a double-blind and placebo-controlled study, 44 healthy subjects aged 23 to 63 years consumed either standard or n-3 LCPUFA-enriched versions of typical processed foods, the latter allowing a target daily consumption of 1 gram n-3 LCPUFA. After six months, peripheral blood leukocyte and subpopulation proportions and numbers were assessed by flow cytometry. Leukocytes were also examined for lymphoproliferation and cytokine production, neutrophil chemotaxis, chemokinesis, bactericidal, adherence and iodination activity. Erythrocytes were analyzed for fatty-acid content. Results : Erythrocyte n-3 LCPUFA levels were higher and absolute leukocyte and lymphocyte numbers were lower in subjects consuming n-3 enriched foods than in controls. There were no changes in the number of neutrophils, monocytes, T cells (CD3+), T-cell subsets (CD4+, CD8+) and B cells (CD19+). However, natural killer (NK) (CD3-CD16+CD56+) cell numbers were lower in n-3 supplemented subjects than in controls and were inversely related to the amount of eicosapentaenoic acid or docosahexaenoic acid in erythrocytes. No significant correlations were found with respect to lymphocyte lymphoproliferation and production of IFN-γ and IL-2, but lymphotoxin production was higher with greater n-3 LCPUFA membrane content. Similarly, neutrophil chemotaxis, chemokinesis, bactericidal activity and adherence did not vary with changes in erythrocyte n-3 LCPUFA levels, but the iodination reaction was reduced with higher n-3 LCPUFA content. Conclusion : The data show that regular long-term consumption of n-3 enriched foods leads to lower numbers of NK cells and neutrophil iodination activity but higher lymphotoxin production by lymphocytes. These changes are consistent with decreased inflammatory reaction and tissue damage seen in patients with inflammatory disorders receiving n-3 LCPUFA supplementation.

The effect of an ultra-endurance running race on mucosal and humoral immune function

There are reports of the effect of endurance exercise on mucosal immune function and of the effect of short duration exercise on humoral immune function. However, little is known of the effect of endurance exercise on humoral immune function and the related risk of infection. This study examined the effects of an ultra-endurance running race on salivary immunoglobulin-A (s-IgA), serum IgA, leukocyte subset concentrations and the incidence of upper respiratory tract infections (URTI). 

Regulation of granulocyte colony-stimulating factor and its receptor in skeletal muscle is dependent upon the type of inflammatory stimulus

The cytokine granulocyte colony-stimulating factor (G-CSF) binds to its receptor (G-CSFR) to stimulate hematopoietic stem cell mobilization, myelopoiesis, and the production and activation of neutrophils. In response to exercise-induced muscle damage, G-CSF is increased in circulation and G-CSFR has recently been identified in skeletal muscle cells. While G-CSF/G-CSFR activation mediates pro- and anti-inflammatory responses, our understanding of the role and regulation in the muscle is limited. The aim of this study was to investigate, in vitro and in vivo, the role and regulation of G-CSF and G-CSFR in skeletal muscle under conditions of muscle inflammation and damage. First, C2C12 myotubes were treated with lipopolysaccharide (LPS) with and without G-CSF to determine if G-CSF modulates the inflammatory response. Second, the regulation of G-CSF and its receptor was measured following eccentric exercise-induced muscle damage and the expression levels we investigated for redox sensitivity by administering the antioxidant N-acetylcysteine (NAC). LPS stimulation of C2C12 myotubes resulted in increases in G-CSF, interleukin (IL)-6, monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-α (TNFα) messenger RNA (mRNA) and an increase in G-CSF, IL-6, and MCP-1 release from C2C12 myotubes. The addition of G-CSF following LPS stimulation of C2C12 myotubes increased IL-6 mRNA and cytokine release into the media, however it did not affect MCP-1 or TNFα. Following eccentric exercise-induced muscle damage in humans, G-CSF levels were either marginally increased in circulation or remain unaltered in skeletal muscle. Similarly, G-CSFR levels remained unchanged in response to damaging exercise and G-CSF/G-CSFR did not change in response to NAC. Collectively, these findings suggest that G-CSF may cooperate with IL-6 and potentially promote muscle regeneration in vitro, whereas in vivo aseptic inflammation induced by exercise did not change G-CSF and G-CSFR responses. These observations suggest that different models of inflammation produce a different G-CSF response.

Ibuprofen ingestion does not affect markers of post-exercise muscle inflammation

PURPOSE: We investigated if oral ingestion of ibuprofen influenced leucocyte recruitment and infiltration following an acute bout of traditional resistance exercise Methods: Sixteen male subjects were divided into two groups that received the maximum over-the-counter dose of ibuprofen (1200mg d(-1)) or a similarly administered placebo following lower body resistance exercise. Muscle biopsies were taken from m.vastus lateralis and blood serum samples were obtained before and immediately after exercise, and at 3 and 24 h after exercise. Muscle cross-sections were stained with antibodies against neutrophils (CD66b and MPO) and macrophages (CD68). Muscle damage was assessed via creatine kinase and myoglobin in blood serum samples, and muscle soreness was rated on a ten-point pain scale.

RESULTS: The resistance exercise protocol stimulated a significant increase in the number of CD66b(+) and MPO(+) cells when measured 3 h post exercise. Serum creatine kinase, myoglobin and subjective muscle soreness all increased post-exercise. Muscle leucocyte infiltration, creatine kinase, myoglobin and subjective muscle soreness were unaffected by ibuprofen treatment when compared to placebo. There was also no association between increases in inflammatory leucocytes and any other marker of cellular muscle damage.

CONCLUSION: Ibuprofen administration had no effect on the accumulation of neutrophils, markers of muscle damage or muscle soreness during the first 24 h of post-exercise muscle recovery.