Leukocyte IL-6 Research Articles

Modulation of gene expression and inflammation but not DNA damage after sevoflurane anesthesia.

This study assessed, for the first time, the expression of the genes hOGG1, TP53, and IL-6 in leukocytes by real-time quantitative polymerase chain reaction in surgical patients before (baseline), during (2 h of anesthesia) and 1 day after sevoflurane anesthesia. Additionally, DNA damage was detected by the comet assay, serum interleukin (IL)-6 was detected by flow cytometry, and differential leukocyte counting was also performed. TP53 and hOGG1 expression was downregulated on the day after anesthesia compared to before anesthesia. However, IL-6 expression did not change, and no DNA damage induction was observed during or after anesthesia. At the systemic level, mild neutrophilia and an increase in IL-6 levels occurred after anesthesia. Our findings suggest that sevoflurane anesthesia downregulates gene expression (hOGG1 and TP53) and contributes to an inflammatory status (increased systemic IL-6 and mild neutrophilia) but is not associated with DNA damage in patients without comorbidities who undergo minor elective surgery.

Lactate production by Staphylococcus aureus biofilm inhibits HDAC11 to reprogram the host immune response during persistent infection

Abstract Staphylococcus aureus (S. aureus) is a leading cause of prosthetic joint infection (PJI) typified by biofilm formation. Our laboratory has identified preferential myeloid-derived suppressor cell (MDSC) recruitment as a critical mechanism for biofilm persistence, as MDSCs are a main source of IL-10. We screened the Nebraska Transposon Mutant Library to identify S. aureus mutants impaired in their ability to trigger MDSC IL-10 production. Significant hits in lactate biosynthesis were identified. A S. aureus Δddh/ldh1/ldh2 mutant, defective in both D- and L-lactate production, decreased leukocyte IL-10 expression and biofilm-associated monocytes were reprogrammed to a pro-inflammatory state, resulting in reduced biofilm burden. Histone acetylation plays a central role in regulating gene expression and histone deacetylase (HDAC) activity was significantly increased in leukocytes recovered from mice infected with Δddh/ldh1/ldh2 compared to WT, resulting in decreased global histone 3 (H3) acetylation at the IL-10 promoter in MDSCs from Δddh/ldh1/ldh2-infected mice. Lactate inhibited HDAC11, resulting in unchecked activity of HDAC6, a positive regulator of Il-10 transcription, and increased IL-10 production. MDSCs and macrophages produced significantly less IL-10 when treated with HDAC6 inhibitor tubastatin A during co-culture with WT but not Δddh/ldh1/ldh2 biofilm. D-lactate was elevated in synovial fluid of patients with PJI compared to aseptic controls and lactate promoted IL-10 production by human monocyte-derived macrophages. Collectively, S. aureus lactate-mediated inhibition of HDAC11 plays a critical role during biofilm infection, leading to epigenetic changes that reprogram the host immune response.

Reactive Oxygen Species Secreted by Leukocytes in Semen Induce Self-Expression of Interleukin-6 and Affect Sperm Quality.

Reproductive tract inflammation is considered an important cause of male infertility. Increased leukocytes in semen can produce many reactive oxygen species (ROS), which affect sperm function. The aim of this study is to identify the main source of ROS in seminal plasma and to assess the effect of ROS on leukocytes. Semen samples (n = 20) with leukocyte concentration >1 × 106 were collected from a male infertility clinic. This study mainly compares the sperm function parameters of the normal group and the semen white blood cell group >1 × 106. The results identified that ROS in semen was closely related to sperm function parameters, and CD45+ leucocytes were the main source of ROS. Compared with the control group, the concentration of IL-2, IL-4, IL-6, IFN-γ, and TNF-α was higher in the experimental group. Leukocytes in semen may regulate the secretion of ROS through the mammalian target of rapamycin (mTOR) pathway. A considerable amount of ROS can upregulate the expression of IL-6 in leukocytes via the nuclear factor kappa-B (NF-kB) pathway.

Lactate production by Staphylococcus aureus biofilm inhibits HDAC11 to reprogramme the host immune response during persistent infection.

Staphylococcus aureus (S. aureus) is a leading cause of biofilm-associated prosthetic joint infection (PJI), resulting in significant disability and prolonged treatment. It is known that host leukocyte IL-10 production is required for S. aureus biofilm persistence in PJI. A S. aureus bursa aurealis Tn library consisting of 1,952 non-essential genes was screened for mutants that failed to induce IL-10 in myeloid-derived suppressor cells (MDSCs), which identified a critical role for bacterial lactic acid biosynthesis. We generated a S. aureus ddh/ldh1/ldh2 triple Tn mutant that cannot produce D- or L-lactate. Co-culture of MDSCs or macrophages with ddh/ldh1/ldh2 mutant biofilm produced substantially less IL-10 compared with wild type S. aureus, which was also observed in a mouse model of PJI and led to reduced biofilm burden. Using MDSCs recovered from the mouse PJI model and in vitro leukocyte-biofilm co-cultures we show that bacterial-derived lactate inhibits histone deacetylase 11 (HDAC11), causing unchecked HDAC6 activity and increased histone 3 acetylation at the Il-10 promoter, resulting in enhanced Il-10 transcription in MDSCs and macrophages. Finally, we show that synovial fluid of patients with PJI contains elevated amounts of D-lactate and IL-10 compared with control subjects, and bacterial lactate increases IL-10 production by human monocyte-derived macrophages.

Staphylococcus aureus biofilm-derived lactate inhibits HDAC11 to augment leukocyte IL-10 production and promote infection persistence

Abstract Staphylococcus aureus (S. aureus) is a leading cause of biofilm-associated prosthetic joint infections (PJIs), where leukocyte IL-10 production is critical for biofilm persistence. We screened a S. aureus mutant library that identified the importance of lactate biosynthetic pathways to elicit IL-10 production. A S. aureus Δddh/ldh1/ldh2 mutant, defective in both D- and L-lactate production, led to significantly decreased leukocyte IL-10 expression and re-programmed biofilm-associated monocytes to a pro-inflammatory state, resulting in reduced biofilm burden. Histone acetylation plays a central role in regulating gene expression, and we found that histone deacetylase (HDAC) activity was increased in Δddh/ldh1/ldh2-infected mice compared to WT concomitant with significant decreases in global histone 3 (H3) acetylation and H3 acetylation at the IL-10 promoter, demonstrating that S. aureus-derived lactate is a HDAC inhibitor. Lactate was found to inhibit HDAC11, which resulted in unregulated HDAC6 activity and increased IL-10 production. MDSCs and macrophages produced significantly less IL-10 in the presence of the HDAC6 inhibitor tubastatin A during co-culture with WT but not Δddh/ldh1/ldh2 biofilms, while HDAC11 KO MDSCs and macrophages co-cultured with Δddh/ldh1/ldh2 biofilms produced significantly more IL-10 compared to WT cells due to unchecked HDAC6 action. D-lactate was elevated in synovial fluid of patients with PJI compared to aseptic controls and lactate also promoted IL-10 production by human monocyte-derived macrophages. Collectively, S. aureus lactate-mediated inhibition of HDAC11 plays a critical role during biofilm infection, leading to epigenetic changes that reprogram the host immune response.

Dogs with osteosarcoma have altered pro‐ and anti‐inflammatory cytokine profiles

BackgroundCurrent advances in immunotherapy are an exciting area of study in canine osteosarcoma (OSA). The objective of this study was to determine the immune response in dogs with osteosarcoma by measuring stimulated leukocyte production of tumor necrosis factor (TNF), interleukin (IL)‐6, IL‐10 and TNF and IL‐6 to IL‐10 ratios.MethodsWhole blood was collected from dogs with osteosarcoma receiving non‐steroidal anti‐inflammatory drugs (NSAIDs, n = 11), dogs with osteosarcoma not receiving NSAIDs (n = 14) and healthy dogs (n = 5).ResultsNo difference in TNF production was found among healthy and OSA dogs regardless of NSAID administration following stimulation with lipopolysaccharide (LPS) (p = .410), lipoteichoic acid (LTA) (p = .693) or PBS (p = .120). Leukocyte IL‐6 production was greater in all dogs with OSA after stimulation with LPS (p = .015), LTA (p = .014) and PBS (p = .034) with no difference between OSA dogs receiving NSAIDs and those not. No differences in IL‐10 were found among healthy controls and dogs with OSA regardless of NSAID use. There was no difference among groups for LPS‐stimulated TNF to IL‐10 ratios (p = .407). For LTA‐stimulated leukocytes, the TNF to IL‐10 ratio was lower in dogs with OSA than in healthy dogs (p = .031) with no difference between OSA NSAID dogs compared to OSA non‐NSAID dogs (p = .059). No differences were found in LPS (p = .310)‐ or LTA (p = .265)‐stimulated leukocyte IL‐6 to IL‐10 production ratios among groups.ConclusionsDogs with osteosarcoma have an altered pro‐ and anti‐inflammatory immunologic profile compared to healthy dogs regardless of NSAID use. Further study is indicated to determine the potential prognostic and therapeutic implications of these findings.

Postprandial inflammatory responses after oral glucose, lipid and protein challenges: Influence of obesity, sex and polycystic ovary syndrome

Most evidence linking the polycystic ovary syndrome (PCOS) with chronic low-grade inflammation has been obtained in the fasting state. We have studied the postprandial inflammatory response to oral glucose, lipid and protein challenges and the possible influences of obesity, sex and PCOS on these responses. On alternate days, we submitted 17 women with PCOS (9 non-obese, 8 obese), 17 control women (9 non-obese, 8 obese) and 19 control men (10 non-obese, 9 obese) to isocaloric (300Kcal) oral macronutrient loads. We assayed serum for TNF-α, IL-6, IL-18, IL-10, pentraxin-3 and galectin-3 concentrations and leukocytes for expression of TNF, IL6, IL10 and their receptors TNFRSF1B, IL6R and IL10RA. Circulating IL-6 levels decreased after glucose and protein ingestion but slightly increased after oral lipid intake. Leukocyte IL6 expression did not change after the ingestion of any macronutrient yet IL6R expression increased during all macronutrient challenges, the largest increase being observed after glucose ingestion. Serum TNF-α similarly decreased during either macronutrient load, whereas TNF expression increased after macronutrient ingestion, the highest increase observed after oral glucose. TNFRSF1B expression also increased after glucose intake but not after lipid or protein ingestion. No global effect of obesity or group on postprandial circulating IL-6, TNF-α, or IL6, IL6R, TNF and TNFRSF1B expression was found. Circulating IL-18 concentrations decreased during all oral challenges, whereas in case of galectin-3 and pentraxin-3 only the protein load caused a reduction in its concentrations. Of the genes studied here, IL10 showed the largest increase in expression throughout all the postprandial curves, particularly after glucose. Obesity blunted the increase in IL10 expression. IL10RA expression decreased after glucose ingestion but remained unchanged during lipid and protein loads. Glucose ingestion, as opposed to lipid and protein intake, results into the largest increase in leukocyte gene expression of inflammatory mediators. The expression of the anti-inflammatory cytokine IL10 was the largest observed here, suggesting a compensatory mechanisms against postprandial inflammation that may be blunted in obesity. However, these responses did not translate into the circulating concentrations of these inflammatory mediators during the immediate postprandial phase.

Identification of immunologic and clinical characteristics that predict inflammatory response to C. Novyi-NT bacteriolytic immunotherapy

BackgroundClostridium novyi-NT (CNV-NT), has shown promise as a bacterolytic therapy for solid tumors in mouse models and in dogs with naturally developing neoplasia. Factors that impact the immunologic response to therapy are largely unknown. The goal of this pilot study was to determine if plasma immune biomarkers, immune cell function, peripheral blood cytological composition and tumor characteristics including evaluation of a PET imaging surrogate of tumor tissue hypoxia could predict which dogs with naturally developing naïve neoplasia would develop an inflammatory response to CNV-NT.ResultsDogs that developed an inflammatory response to CNV-NT had a higher heart rate, larger gross tumor volume, greater tumor [64Cu]ATSM SUVMax, increased constitutive leukocyte IL-10 production, more robust NK cell-like function and greater peripheral blood lymphocyte counts compared to dogs that did not develop an inflammatory response to CNV-NT. Of these, unstimulated leukocyte IL-10 production, heart rate, and gross tumor volume appeared to be the best predictors of which dogs will develop an inflammatory response to CNV-NT.ConclusionsDevelopment of inflammation in response to CNV-NT is best predicted by pretreatment unstimulated leukocyte IL-10 production, heart rate, and gross tumor volume.

Effects of parenteral fish oil on plasma nonesterified fatty acids and systemic inflammatory mediators in dogs following ovariohysterectomy.

To evaluate the effects of intravenous (IV) infusion of fish oil (FO) emulsion following ovariohysterectomy (OVH) on inflammatory mediators and plasma omega-3 nonesterified fatty acids (NEFA) concentrations in dogs. Prospective clinical study. University teaching hospital. Twenty-nine privately owned dogs undergoing routine OVH. Postoperative 3-hour IV infusion of saline (n = 9), FO (Omegaven, n = 10), or soybean oil (SO, intralipid, n = 10) emulsion and blood collected before, 5 and 24 hours following OVH for plasma NEFA and RBC membrane fatty acids (FAs) concentrations, leukocyte cytokine production capacity, and C-reactive protein (CRP) measurement. Plasma omega-3 NEFA, eicosapentaenoic acid, docosahexaenoic acid, and total long-chain omega-3 FA significantly increased shortly after FO infusion (8.8±3.3 μM, 13.6±5.6 μM, and 25.1±9.6 μM, respectively) compared to SO (0.7±0.9, 2.3±1.8, and 4.2±3.0 μM, respectively) and saline infusion (1.6±2.5, 2.6±3.1, and 5.9±6.4 μM, respectively). Plasma CRP concentration significantly increased after OVH, but with no significant group differences. A weak negative correlation occurred between post-OVH CRP and postinfusion total long-chain omega-3 FA concentrations (r2 = 0.21, P = 0.014). Stimulated leukocyte interleukin (IL) 6 production capacity increased (P = 0.001) after OVH in all groups; SO infusion resulted in reduced leukocyte IL-6 production capacity (1048.1±277.7 pg/mL) compared to FO (1299.9±302.1 pg/mL, P = 0.048) and saline infusions (1499.0±363.1 pg/mL, P = 0.01). No significant group difference was observed in leukocyte IL-10 and tumor necrosis factor α production capacities. Postoperative administration of FO emulsion increases plasma omega-3 NEFA concentrations promptly, but does not significantly attenuate CRP production or leukocyte cytokine production capacity. FO infusion at the dosage used in the present study can be safely used in dogs, but it was not clearly beneficial in decreasing post-OVH indices of inflammation.

Oral warfarin affects peripheral blood leukocyte IL-6 and TNFα production in rats

Warfarin is a Vitamin K (VK) antagonist that affects Vitamin K-dependent (VKD) processes, including blood coagulation, as well as processes unrelated to hemostasis such as bone growth, calcification, and growth of some cell types. In addition, warfarin exerts influence on some non-VKD-related activities, including anti-tumor and immunomodulating activity. With respect to the latter, both immune stimulating and suppressive effects have been noted in different experimental systems. To explore the in vivo immunomodulatory potential of warfarin on one type of activity (i.e., cytokine production) in two different immune cell populations (i.e., mononuclear or polymorphonuclear cells), effects of subchronic oral warfarin intake in rats on pro-inflammatory cytokine (i.e., TNFα, IL-6) production by peripheral blood mononuclear and polymorphonuclear cells (granulocytes) was examined. Differential effects of warfarin intake on TNFα and IL-6 were noted, depending on the type of peripheral blood leukocytes and on the cytokine examined. Specifically, a lack of effect on TNFα and a priming of IL-6 production by mononuclear cells along with a decrease in TNFα and a lack of effect on IL-6 in polymorphonuclear cells were seen in warfarin-exposed hosts. The cell- and cytokine-dependent effects from subchronic oral warfarin intake on peripheral blood leukocytes demonstrated in this study could, possibly, differentially affect reactions mediated by these cells. Ultimately, the observed effects in rats might have implications for those humans who are on long-term/prolonged warfarin therapy.

The effect of dietary fatty acids on post-operative inflammatory response in a porcine model

The potential anti-inflammatory effects of dietary fish oil (FO) have been studied in numerous clinical trials. However, variation in lifestyle and morbidity among patients can be difficult to control. In the present study, the impact of a 3-week dietary pre-treatment with 10% (w/w) FO (n 28), sunflower oil (SO, n 28), or animal fat (AF, n 28) was evaluated with respect to post-operative responses in inflammatory markers in a porcine model on aortic vascular prosthetic graft infection. In the early post-operative period (0 < day ≤ 3), FO suppressed whole blood IL-8 and tumor necrosis factor-α responsiveness to LPS stimulation, decreased peripheral leukocyte IL-8 mRNA abundance, reinforced an increase in total leukocyte count, and counteracted a decrease in mononuclear leukocyte count compared with SO. In the late post-operative period (3 < day ≤ 14), FO increased total leukocyte count and showed higher maximum CRP and haptoglobin concentrations compared with SO. Compared with AF, FO decreased peripheral leukocyte IL-8 mRNA abundance in the early post-operative period, and increased total leukocyte count and maximum CRP concentration in the late post-operative period. In conclusion, the post-operative response in a number of inflammatory markers was affected by FO, and this was most apparent compared with SO.

β-Glucan, Immune Function, and Upper Respiratory Tract Infections in Athletes

This study investigated the effects of oat beta-glucan (BG) supplementation on chronic resting immunity, exercise-induced changes in immune function, and self-reported upper respiratory tract infection (URTI) incidence in human endurance athletes. Trained male cyclists were randomized to BG (N = 19) or placebo (P; N = 17) groups and under double-blind procedures received BG (5.6 g x d(-1)) or P beverage supplements for 2 wk before, during, and 1 d after a 3-d period in which subjects cycled for 3 h x d(-1) at approximately 57% maximal watts. URTI symptoms were monitored during BG supplementation and for 2 wk afterward. Blood samples were collected before and after 2 wk of supplementation (both samples, 8:00 a.m.), immediately after the 3-h exercise bout on day 3 (6:00 p.m.), and 14 h after exercise (8:00 a.m.) and were assayed for natural killer cell activity (NKCA), polymorphonuclear respiratory burst activity (PMN-RBA), phytohemagglutinin-stimulated lymphocyte proliferation (PHA-LP), plasma interleukin 6 (IL-6), IL-10, IL-1 receptor agonist (IL-1ra), and IL-8, and blood leukocyte IL-10, IL-8, and IL-1ra mRNA expression. Chronic resting levels and exercise-induced changes in NKCA, PMN-RBA, PHA-LP, plasma cytokines, and blood leukocyte cytokine mRNA did not differ significantly between BG and P groups. URTI incidence during the 2-wk postexercise period did not differ significantly between groups. An 18-d period of BG versus P ingestion did not alter chronic resting or exercise-induced changes in immune function or URTI incidence in cyclists during the 2-wk period after an intensified exercise.

Effects Of β-Glucan On Immune Function And Upper Respiratory Tract Infections In Endurance Athletes

Animal data indicate that oat β-glucan (BG) supplements offset the increased risk of upper respiratory tract infection (URTI) associated with exercise stress through augmentation of neutrophil and macrophage function. PURPOSE: This study investigated the effects of BG supplementation on chronic resting and exercise-induced changes in immune function and URTI incidence in human endurance athletes. METHODS: Trained male cyclists were randomized to BG (N=19) or placebo (P) (N=17) groups and under double blind procedures received BG (5.6 grams/day) or P for two weeks prior to and during a 3-d period in which subjects cycled for 3 h/day at ~57% Wattsmax. URTI symptoms were monitored for two weeks before and two weeks after the 3-day period of intensified exercise. Blood samples were collected before and after two weeks supplementation (both samples, 8:00 am), immediately after the 3 h exercise bout on day 3 (6:00 pm), and 14 h post-exercise (8:00 am), and assayed for natural killer cell activity (NKCA), polymorphonuclear respiratory burst activity (PMN-RBA), PHA-stimulated lymphocyte proliferation (PHA-LP), plasma IL-6, IL-10, IL-1ra, and IL-8, and blood leukocyte IL-10, IL-8, and IL-1ra mRNA expression. RESULTS: Chronic resting levels and exercise-induced changes in NKCA, PMN- RBA, PHA-LP, plasma cytokines, and blood leukocyte cytokine mRNA did not differ significantly between BG and P groups. URTI incidence rates during the two week period following intensified exercise did not differ significantly between groups (9/19 or 47% for BG and 6/17 or 35% for P, Chi-square = 0.16, P=0.693). CONCLUSION: A 17-day period of BG vs. P ingestion did not alter chronic resting or exercise-induced changes in immune function, or URTI incidence in cyclists during the 2-week period following intensified exercise. Funded by a grant from the Gatorade Sports Science Institute.

Quercetin Ingestion Does Not Alter Cytokine Changes in Athletes Competing in the Western States Endurance Run

The purpose of this study was to measure the influence of quercetin on plasma cytokines, leukocyte cytokine mRNA, and related variables in ultramarathoners competing in the 160-km Western States Endurance Run (WSER). Sixty-three runners were randomized to quercetin and placebo groups and under double-blinded methods ingested 1000 mg/day quercetin for 3 weeks before the WSER. Thirty-nine of the 63 subjects (n = 18 for quercetin, n = 21 for placebo) finished the race and provided blood samples the morning before the race and 15-30 min postrace. Significant prerace to postrace WSER increases were measured for nine proinflammatory and anti-inflammatory plasma cytokines, cortisol (quercetin = 94%, placebo = 96%), serum C-reactive protein (CRP) (mean +/- SE absolute increase, quercetin = 31.8 +/- 4.2, placebo = 38.2 +/- 5.0 mg/L), and creatine kinase (CK) (quercetin = 21,575 +/- 3,977, placebo = 19,455 +/- 3,969 U/L), with no significant group differences. Interleukin-6 (IL-6) mRNA did not change post-WSER, with a significant decrease measured for leukocyte IL-8 mRNA (0.21 +/- 0.03-fold and 0.25 +/- 0.04-fold change from rest, quercetin and placebo, respectively) and significant increases for IL-1Ra mRNA (1.43 +/- 0.18-fold and 1.40 +/- 0.16-fold change, quercetin and placebo, respectively) and IL-10 mRNA (12.9 +/- 3.9-fold and 17.2 +/- 6.1-fold change, quercetin and placebo, respectively), with no significant differences between groups. In conclusion, quercetin ingestion (1 g/day) by ultramarathon athletes for 3 weeks before a competitive 160-km race significantly increased plasma quercetin levels but failed to attenuate muscle damage, inflammation, increases in plasma cytokine and hormone levels, and alterations in leukocyte cytokine mRNA expression.

Quercetin's influence on exercise-induced changes in plasma cytokines and muscle and leukocyte cytokine mRNA

Trained male cyclists (n = 40) ingested quercetin (Q; n = 20) (1,000 mg/day) or placebo (P; n = 20) supplements under randomized, double-blinded methods for 3 wk before and during a 3-day period in which subjects cycled for 3 h/day at approximately 57% maximal work rate. Blood samples were collected before and after each exercise session and assayed for plasma IL-6, IL-10, IL-1ra, IL-8, TNF-alpha, and monocyte chemoattractant protein 1, and leukocyte IL-10, IL-8, and IL-1ra mRNA. Muscle biopsies were obtained before and after the first and third exercise sessions and assayed for NF-kappaB and cyclooxygenase-2 (COX-2), IL-6, IL-8, IL-1beta, and TNF-alpha mRNA. Postexercise increases in plasma cytokines did not differ between groups, but the pattern of change over the 3-day exercise period tended to be lower in Q vs. P for IL-8 and TNF-alpha (P = 0.094 for both). mRNA increased significantly postexercise for each cytokine measured in blood leukocyte and muscle samples. Leukocyte IL-8 and IL-10 mRNA were significantly reduced in Q vs. P (interaction effects, P = 0.019 and 0.012, respectively) with no other leukocyte or muscle mRNA group differences. Muscle NF-kappaB did not increase postexercise and did not differ between Q and P. Muscle COX-2 mRNA increased significantly postexercise but did not differ between Q and P. In summary, 1 g/day quercetin supplementation by trained cyclists over a 24-day period diminished postexercise expression of leukocyte IL-8 and IL-10 mRNA, indicating that elevated plasma quercetin levels exerted some effects within the blood compartment. Quercetin did not, however, influence any of the muscle measures, including NF-kappaB content, cytokine mRNA, or COX-2 mRNA expression across a 3-day intensified exercise period.

Quercetin Ingestion Does Not Alter Cytokine Changes In Athletes Competing in the Western States Endurance Run

PURPOSE: To measure the influence of the antioxidant flavonol molecule, quercetin (Q), on plasma cytokines, leukocyte cytokine mRNA, plasma cortisol, muscle damage, C-reactive protein (CRP), and delayed onset of muscle soreness (DOMS) in ultra-marathoners competing in the 160-km Western States Endurance Run (WSER). METHODS: Sixty-three runners were randomized to Q and placebo (P) groups, and under double blinded methods ingested 1000 mg/day Q for 3 wks before, during, and 2 wks after the WSER. Thirty-nine of the 63 subjects (N=18 for Q, N=21 for P) finished the race, provided blood samples the morning before the race and 15–30 min post-race, and recorded DOMS during the week after the race using a 10-point Likert scale. RESULTS: Significant pre-to-post WSER increases were measured for 10 pro- and anti-inflammatory plasma cytokines, cortisol (Q=94%, P=96%), serum CRP (absolute increase, Q=318 and P=381 mg/L), and creatine kinase (CK) (absolute increase, Q=21574 and P=19472 U/L), with no significant group differences. DOMS peaked the day after the WSER (6.8±0.4 and 7.3±0.4 for Q and P, respectively, P=0.377) with no group differences seen during the 7-day post-WSER period. IL-6 mRNA did not change post-WSER, with a decrease measured for leukocyte IL-8 mRNA (0.21 and 0.25 fold change from rest, Q and P respectively), and increases for IL-1ra and IL-10 mRNA (12.9 and 17.2 fold change, respectively), with no significant group differences. CONCLUSION: Q supplementation for 3 wks before and 2 wks after the WSER had no effect on increases in plasma levels for 10 cytokines, cortisol, serum CRP, changes in leukocyte mRNAfor IL-8, IL-1ra, IL-10, and IL-6, muscle damage, and DOMS.

Blood Leukocyte mRNA Expression for IL-10, IL-1Ra, and IL-8, but Not IL-6, Increases After Exercise

The primary purpose of this project was to study exercise-induced leukocyte cytokine mRNA expression. Changes in plasma cytokine levels and blood leukocyte mRNA expression for interleukin-6 (IL-6), IL-8, IL- 10, and IL-1 receptor antagonist (IL-1Ra) were measured in 12 athletes following 2 h of intensive cycling ( approximately 64% Watts(max)) while ingesting a carbohydrate or placebo beverage (randomized and double blinded). Blood samples were collected 30 min preexercise and immediately and 1 h postexercise. Carbohydate compared with placebo ingestion attenuated exercise-induced changes in plasma cortisol (8.8% vs. 62%, respectively), epinephrine (-9.2% vs. 138%), IL-6 (10-fold vs. 40-fold), IL-10 (8.9-fold vs. 26-fold, and IL-1Ra (2.1-fold vs. 5.6-fold). Significant time effects were measured for blood leukocyte IL-8 (2.4-fold increase 1 h postexercise), IL-10 (2.7-fold increase), IL-1Ra (2.2-fold increase), and IL-6 (0.8-fold decrease) mRNA content, with no significant differences between Cho and Pla test conditions. In summary, gene expression for IL-8, IL-10, and IL-1Ra, but not IL-6, is increased in blood leukocytes taken from athletes following 2 h of intensive cycling and is not influenced by carbohydrate compared with placebo ingestion. mRNA expression was high enough to indicate a substantial contribution of blood leukocytes to plasma levels of IL-8, IL-10, and IL-1Ra during prolonged exercise.

Influence of Carbohydrate/Placebo On Immunity Following 2-h Cycling With or Without Rest Intervals

PURPOSE: The purpose of this project was to study the effect of carbohydrate compared to placebo ingestion on immune changes following 2 hours of intensive cycling with or without rest intervals. METHODS: Trained cyclists (N=12) functioned as their own controls during four test sessions that were separated by 1–2 weeks and randomized to control for an order effect. Subjects cycled for 2.0 h at ∼60% Wattsmax continuously (C) or with 3-min rest intervals (R) interspersed every 10 min (2.6 h total time) while receiving 4 ml kg−1.15 min-1 carbohydrate (6%) (Cho) or placebo (Pla) beverages (thus CCho, CPla, RCho, RPla). Blood samples were collected 30 min pre-exercise, and immediately and 1-h post-exercise. Immune and hormonal measures included determination of leukocyte subset counts, plasma IL-6, IL-10, IL-1ra, IL-8, cortisol, glucose, and insulin, PHA-induced lymphocyte proliferation, and natural killer cell activity (NKCA). Blood leukocyte relative gene expression was measured for four cytokines (IL-6, IL-8, IL-10, IL-1ra) using real-time quantitative RT-PCR. RESULTS: Exercise-induced immune and hormonal changes did not differ between C and R trials. Cho compared to Pla ingestion attenuated exercise-induced changes in blood neutrophil, monocyte, T cell, and NK cell counts, plasma cortisol and insulin, plasma IL-6, IL-10, and IL-1ra, and PHA-induced lymphocyte proliferation, but not NKCA (interaction effect, P=0.134). Significant time effects were measured for leukocyte IL-10 (increase), IL-1ra (increase), and IL-6 (decrease) mRNA content with no significant differences measured when comparing C or R exercise modes or Cho and Pla test conditions. The patterns of change in leukocyte IL-8 mRNA did not differ between Cho and Pla, but increased during C and decreased during R exercise trials (exercise mode × time interaction effect, p < 0.001). CONCLUSIONS: Most measured immune and hormonal changes induced by intense and prolonged exercise were attenuated when cyclists ingested Cho compared to Pla beverages, but were largely unaffected when athletes were allowed to rest 3 min every 10 min of exercise.

Enhanced glucocorticoid sensitivity of cytokine release from circulating leukocytes stimulated with lipopolysaccharide in healthy male smokers

Smoking is a strong cardiovascular risk factor that promotes inflammation. The source of elevated pro-inflammatory cytokines in the circulation of smokers is not fully understood. We investigated the release of pro-inflammatory cytokines from circulating leukocytes stimulated with lipopolysaccharide (LPS) and its inhibition by glucocorticoids in smokers and non-smokers. Ninety-three middle-aged apparently healthy men were categorized as smokers (>10 cigarettes/day; n=41) or life-long non-smokers ( n=52). Peripheral cortisol was assessed from overnight urine. C-reactive protein (CRP) and tumor necrosis factor (TNF)-α were measured in plasma. LPS-stimulated interleukin (IL)-6 and TNF-α release from harvested circulating leukocytes were assessed using an in vitro whole blood assay with and without co-incubation of increasing concentrations of either dexamethasone or hydrocortisone. Glucocorticoid sensitivity was defined as the concentration of glucocorticoids required that inhibits LPS-stimulated cytokine release by 50%. Smokers had higher CRP levels ( p=.005) and a trend for higher basal TNF-α levels ( p<.07), and they also showed lower IL-6 and TNF-α release after LPS-stimulation than non-smokers ( p’s < .001). While peripheral cortisol concentration showed no significant group difference, inhibition of LPS-stimulated leukocyte IL-6 and TNF-α release by either glucocorticoid was enhanced in smokers as compared to non-smokers ( p’s < .022). The finding suggests that, in spite of a low-grade systemic inflammation, smokers have decreased LPS-stimulated cytokine release from circulating leukocytes and greater glucocorticoid sensitivity of this cytokine release than non-smokers. Circulating leukocytes unlikely contribute to the elevated pro-inflammatory cytokine levels in the blood of smokers.

Resident cardiac mast cells degranulate and release preformed TNF-alpha, initiating the cytokine cascade in experimental canine myocardial ischemia/reperfusion.

Neutrophil-induced cardiomyocyte injury requires the expression of myocyte intercellular adhesion molecule (ICAM)-1 and ICAM-1-CD11b/CD18 adhesion. We have previously demonstrated interleukin (IL)-6 activity in postischemic cardiac lymph; IL-6 is the primary stimulus for myocyte ICAM- 1 induction. Furthermore, we found that induction of IL-6 mRNA occurred very early on reperfusion of the infarcted myocardium. We hypothesized that the release of a preformed upstream cytokine induced IL-6 in leukocytes infiltrating on reperfusion. Constitutive expression of TNF-alpha and not IL-1beta was demonstrated in the normal canine myocardium and was localized predominantly in cardiac mast cells. Mast cell degranulation in the ischemic myocardium was documented by demonstration of a rapid release of histamine and TNF-alpha in the cardiac lymph after myocardial ischemia. Histochemical studies with FITC-labeled avidin demonstrated degranulating mast cells only in ischemic samples of canine myocardium. Immunohistochemistry suggested that degranulating mast cells were the primary source of TNF-alpha in the ischemic myocardium. In situ hybridization studies of reperfused myocardium localized IL-6 mRNA in infiltrating mononuclear cells and in mononuclear cells appearing in the postischemic cardiac lymph within the first 15 minutes of reperfusion. Furthermore, isolated canine mononuclear cells incubated with postischemic cardiac lymph demonstrated significant induction of IL-6 mRNA, which was partially blocked with a neutralizing antibody to TNF-alpha. Cardiac mast cells degranulate after myocardial ischemia, releasing preformed mediators, such as histamine and TNF-alpha. We suggest that mast cell-derived TNF-alpha may be a crucial factor in upregulating IL-6 in infiltrating leukocytes and initiating the cytokine cascade responsible for myocyte ICAM-1 induction and subsequent neutrophil-induced injury.