Constitutive pro- and anti-inflammatory cytokine and growth factor response to exercise in leukocytes.

Leukocytosis following exercise is a well-described phenomenon of stress/inflammatory activation in healthy humans. We hypothesized that, despite this increase in circulating inflammatory cells, exercise would paradoxically induce expression of both pro- and anti-inflammatory cytokines and growth factors within these cells. To test this hypothesis, 11 healthy adult men, 18-30 yr old, performed a 30-min bout of heavy cycling exercise; blood sampling was at baseline, end-exercise, and 60 min into recovery. The percentage of leukocytes positive for intracellular cytokines and growth factors and mean fluorescence intensity was obtained by flow cytometry. Proinflammatory cytokines (IL-1alpha, IL-2, IFN-gamma, and TNF-alpha), a pleiotropic cytokine (IL-6), and anti-inflammatory cytokines and growth factors [IL-4, IL-10, growth hormone (GH), and IGF-I] were examined. Median fluorescence intensity was not affected by exercise; however, we found a number of significant changes (P < 0.05 by mixed linear model and modified t-test) in the numbers of circulating cells positive for particular mediators. The pattern of expression reflected both pro- and anti-inflammatory functions. In T-helper lymphocytes, TNF-alpha, but also IL-6, and IL-4 were significantly increased. In monocytes, both IFN-gamma and IL-4 increased. B-lymphocytes positive for GH and IGF-I increased significantly. GH-positive granulocytes also significantly increased. Collectively, these observations indicate that exercise primes an array of pro- and anti-inflammatory and growth factor expression within circulating leukocytes, perhaps preparing the organism to effectively respond to a variety of stressors imposed by exercise.     

Inhibitory action of azelastine on cytokine production from human peripheral blood leukocytes in vitro

Effects of azelastine (AZ) on human peripheral blood leukocytes (PBL) in culture were examined. Addition of 10.0 μg/ml of AZ resulted in a marked inhibition of PBL blastic activity, but lower concentrations (1.0 and 0.5 μg/ml) had no demonstrable suppressive effects on the activation. Interleukin (IL)-2, IL-3 and IL-4 production from PBL in response to Concanavalin A stimulation was also strongly suppressed when the cells were cultured in the presence of AZ. This suppression was observed even when lower concentrations (1.0 and 0.5 μg/ml) of AZ were added to cell cultures     

Pro- versus anti-inflammatory cytokine profile in African children with acute oro-facial noma (cancrum oris, noma)

Fresh noma is a severe orofacial necrosis with an astonishingly rapid development. It is seen mainly in malnourished children less than 4 years old from developing countries. Cytokines play a central role in oral mucosal inflammation. We therefore studied the relevance of circulating cytokines to noma, and the key microorganisms associated with the lesion. Nigerian village children with acute noma (n=68) and their neighborhood village (n=63) as well as urban (n=45) counterparts of comparable age and free of overt infections were evaluated for serum cytokine levels by ELISA. Oral bacteria were studied by polymerase chain reaction. Evaluation of random cases of the village and noma children showed marked depletion (p<0.05 or 0.001) of the plasma antioxidant micronutrients (retinol, ascorbic acid, zinc) as well as albumin and blood hemoglobin in the latter, relative to the former group. Concentrations of the circulating, pro-inflammatory cytokines (IL-18, IL-6, IL-12, IL-8, IFN-gamma) and the soluble inhibitors (TNFR-p55, TNFR-p75 and IL-1ra) were significantly higher (p<0.01 or 0.001) in noma children than in the healthy urban children, but less so when compared to their neighborhood village counterparts. The increase in levels of the anti-inflammatory/regulatory cytokines (IL-4, IL-10 and TGF-beta) was less marked relative to the pro-inflammatory cytokines. Bacteria observed at the highest frequencies in noma lesions were P. intermedia (83%), T. forsythensis (83%), P. gingivalis (50%), C. rectus (50%) and T. denticola (50%). We conclude that noma is an immunopathological response to potent bacterial factors resulting in uncontrolled production of cytokines and possibly other, still unknown, inflammatory mediators.     

Colony formation inhibition in human bone marrow stromal cells exposed to a factor formed in vitro by peripheral blood leukocytes]

Addition of human peripheral blood leukocytes or a medium in which the leukocytes were cultivated to the monolayer culture of human bone marrow produced an inhibitory action of the growth of the fibroblast colonies. This effect was not associated with the immunological incompatibility of the cells in a culture--autologous blood cells produced the same action as the heterologous ones. The factor inhibiting the fibroblast growth failed to depress the growth of other cells, of macrophages in particular.     

Quantitative and qualitative assessment of serum- and inflammatory mediator-induced migration of carp (Cyprinus carpio) head kidney neutrophils in vitro

A quantitative transmigration system, permitting the harvest of transmigrated cells for further analysis, was used to study carp head kidney (HK) granulocyte migration in vitro. Pooled carp serum and leukotriene B4 (LTB-4), but not recombinant human C-X-C chemokine ligand 8 (rhCXCL8), recombinant human complement component 5a (rhC5a) or N-formyl-methionyl-leucyl-phenylalanine (FMLP) induced strong migration (up to 70%) of carp HK granulocytes. The transmigrated cells were viable (>or=96%) and uniform (purity >or=97%). After serum- as well as LTB-4-induced transmigration granulocytes produced the same amounts of reactive oxygen species (ROS) as non-migrated cells in HK cell suspension. Their morphology, staining characteristics and flow cytometric scatter characteristics, plus their ability to produce ROS characterised the transmigrated granulocytes as neutrophils. The quantitative transmigration system described here could also serve as an excellent tool for the selective attraction and isolation of highly purified carp neutrophils from HK cell suspensions.     

Stress proteins expression in rat kidney and liver chronically exposed to aluminium sulphate

Aluminium (Al) is the third most widespread metal in the environment. It is toxic for the brain, bone and haematological system but unfortunately very little data exist for other organs. Stress proteins are induced or enhanced against metal toxicity with an essential role in the recovery of organules and other cellular proteins. This immunohistochemical study was performed to analyze the distribution of three stress proteins (HSP25, HSP72, GRP75) in rat kidney and liver orally exposed to Al sulphate daily for 3 and 6 months. Al-induced alterations were further studied by histopathology (H-E, PAS, Perl's, Masson) and ultrastructural morphometry. In the kidney: HSP25 was enhanced in proximal tubules after 6 months Al-exposure when abnormal brush borders were observed; HSP72 was induced in proximal tubules only after long Al-treatment; GRP75 was raised in midcortical area sometimes within nuclei. Furthermore, lysosomal and lipofuscins densities increased in the juxtamedullary tubules after 3 months Al exposure with respect to controls. In the liver: Perl's-positive deposits and fibrosis became evident after Al treatment. HSP25 was very weak; HSP72 focal in pericentral hepatocytes at 3 months and induced also in Kupffer cells at 6 months; GRP75 diffuse in periportal hepatocytes and non parenchymal cells at 6 months. Prolonged Al exposure stimulated stress proteins strictly organ-dependently in the rat. Their distribution in kidney and liver seems related to cumulative sublethal effects induced by metal and could be a sensitive index of Al susceptibility of these organs.     

Stress-related hormones modulate cytokine expression in the head kidney of gilthead seabream (Sparus aurata)

Neuro-endocrine and immune systems closely interact in fish, and their regulation is crucial for the maintenance of good health of cultured fish. We have used the seabream head kidney to study whether stress-related hormones can modulate the immune response. For this purpose, the effects of adrenaline, adrenocorticotropic hormone (ACTH) and cortisol on the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and the anti-inflammatory cytokine TGF-β1 were determined by means of quantitative real-time PCR on isolated head kidney cells. ACTH (150 ng mL⁻¹) caused an acute increase of TNF-α and IL-6 mRNA levels as well as an inhibition of IL-1β expression. The expression of the anti-inflammatory cytokine TGF-β1 was also increased, although in a lower extent. Adrenaline (1 μM) early effects were only clear inhibiting IL-1β expression but not TNF-α, IL-6 or TGF-β1 mRNA levels, while a longer exposure to the hormone inhibited all cytokines. Moreover, cortisol (50 and 100 ng mL⁻¹) reduced the expression of all cytokines in a dose-dependent manner. Bacterial lipopolysaccharide (LPS) stimulated IL-1β expression and inhibited that of the anti-inflammatory TGF-β1, although it was ineffective on TNF-α and IL-6. In addition, adrenaline and cortisol decreased the LPS-stimulated IL-1β expression, further demonstrating their previously reported anti-inflammatory effects. The combination of ACTH and LPS, on the other hand, did not affect LPS-stimulated IL-1β expression but was effective increasing TNF-α expression. Taking all these results in consideration, we conclude that the expression of pro- and anti-inflammatory cytokines in the seabream head kidney is highly influenced by stress-related hormones, thus indicating an important role for the endocrine system in the modulation of the immune response in teleost fish.     

Cardio-respiratory function in carp exposed to environmental nitrite

Continuous monitoring of heart rate, breathing episodes and blood pressure showed that the cardio-respiratory response of carp exposed to nitrite (water concentration, 1 mmol l⁻¹) changes with length of exposure. The animals developed a severe methaemoglobinaemia over the first 24 h of nitrite exposure. The minor changes in plasma HCO₃⁻ and lactate concentration, suggest that the observed hyperventilatory response was sufficient to maintain aerobic metabolism throughout most of the body during this time. During the second 24-h period, the rate of breathing increased further and short periods of bradycardia and hypotension were seen. Over this latter period, the animals increased their use of anaerobic metabolism as illustrated by the mean 48 h blood lactate concentration of 4.8mmol 1⁻¹, a greater than 10-fold increase over pre-exposure values. The increase in blood lactate was accompanied by the predicted metabolic acidosis, however, an alkalosis of respiratory origin and buffering combined to keep the plasma pH absolutely stable throughout the study. This study shows that as the blood oxygen supply is reduced through the development of methaemoglobinaemia, cardio-respiratory compensation by the carp is probably adequate to maintain tissue oxygenation for short periods of nitrite exposure. However, as nitrite exposure proceeds past 24 h, the animals progress into a positive feedback cycle where the high cost of additional ventilation rapidly accelerates their oxygen deficit which cannot be repaid, because <25% of their haemoglobin is available for oxygen binding. Additionally, our data demonstrate a circadian rhythm of physiological response to nitrite and contradict the hypothesis that catecholamine release promotes CO₂ retention in water breathing animals.     

Vascularization and related distribution of leucocytes in carp, Cyprinus carpio L., head kidney

The distribution of lymphoid cells in the carp head kidney was investigated in relation to the vascular system. Blood vessels in the head kidney were histologically identified into arteries, sinusoids and two types of veins: renal veins and portal, which were distinguished by India ink injection into the caudal vein. By histological and histoplanimetrical observations it was found that the head kidney contained a number of lymphoid cells, which mainly aggregate around the connections between the portal veins and sinusoids, and that the cellular density of the aggregations was higher than in the thymus.
Pigment-containing cell clusters were also observed around these connections. This arrangement of the blood vessels suggests that it is one of the structures able to trap foreign materials, and the occurrence of the lymphoid clusters around the portal veins is a phylogenetic sign of the morphological division between granulopoietic and lymphatic tissues in the carp head kidney.     

Evaluation of level of DNA damage in blood leukocytes of non-diabetic and diabetic rat exposed to cigarette smoke

The objective of the present study was to use the comet assay to evaluate the steady-state level of DNA damage in peripheral blood leukocytes from diabetic and non-diabetic female Wistar rats exposed to air or to cigarette smoke. A total of 20 rats were distributed into four experimental groups (n=5 rats/group): non-diabetic (control) and diabetic exposed to filtered air; non-diabetic and diabetic exposed to cigarette smoke. A pancreatic beta (beta)-cytotoxic agent, streptozotocin (40 mg/kg b.w.) was used to induce experimental diabetes in rats. Rats placed into whole-body exposure chambers were exposed for 30 min to filtered air (control) or to tobacco smoke generated from 10 cigarettes, twice a day, for 2 months. At the end of the 2-month exposure period, each rat was anesthetized and humanely killed to obtain blood samples for genotoxicity analysis using the alkaline comet assay. Blood leukocytes sampled from diabetic rats presented higher DNA damage values (tail moment=0.57+/-0.05; tail length=19.92+/-0.41, p<0.05) compared to control rats (tail moment=0.34+/-0.02; tail length=17.42+/-0.33). Non-diabetic (tail moment=0.43+/-0.04, p>0.05) and diabetic rats (tail moment=0.41+/-0.03, p>0.05) exposed to cigarette smoke presented non-significant increases in DNA damage levels compared to control group. In conclusion, our data show that the exposure of diabetic rats to cigarette smoke produced no additional genotoxicity in peripheral blood cells of female Wistar rats.     

The effect of pseudomonas exotoxin A on cytokine production in whole blood exposed to Pseudomonas aeruginosa

To determine the effect of Pseudomonas aeruginosa exotoxin A (P-ExA) on cytokine production, we studied cytokine release induced by heat-killed P. aeruginosa (HKPA) in human whole blood in the presence or absence of P-ExA. P-ExA (0.01-1 microgram ml(-1)) caused a dose-dependent decrease in HKPA-induced production of tumor necrosis factor alpha (TNF), interleukin (IL-) 10, IL-6 and IL-8 (all P<0.05). P-ExA-induced inhibition of IL-10, IL-6 and IL-8 release was not dependent on reduced TNF concentrations, since the relative attenuation of the production of these cytokines was similar in the presence or absence of a neutralizing anti-TNF antibody. The effect of P-ExA on cytokine production may offer a disadvantage to the host with respect to clearance of the infection.     

Renal response to hypoxia in carp,Cyprinus carpio: Changes in glomerular filtration rate,urine and blood properties and plasma catecholamines of carp exposed to hypoxic conditions

• 1.1. Changes in glomerular nitration rate (GFR), urine and blood properties and plasma catecholamines of carp were investigated during and following hypoxia.
• 2.2. GFR and urine flow decreased with increased urinary concentrations of bio-components, except protein, in the course of hypoxia.
• 3.3. Decreases in blood pH, and increases in haematocrit value and plasma K⁺, Ca²⁺, Mg²⁺, inorganic phosphate (P_i), ammonia, lactic acid and catecholamines (CAs) were observed as hypoxia progressed.
• 4.4. Increased GFR and urine flow, and higher values for urinary components, except protein, compared with those of the control were found in the initial post-stress stage.
• 5.5. The possible significance of increased plasma CAs in relation to changes in renal function in hypoxic carp is discussed.