Differential regulation of cytokine and chemokine production in lipopolysaccharide-induced tolerance and priming

LPS pretreatment of human pro-monocytic THP-1 cells induces tolerance to secondary LPS stimulation with reduced TNFalpha production. However, secondary stimulation with heat-killed Staphylococcus aureus (HKSa) induces priming as evidenced by augmented TNFalpha production. The pro-inflammatory cytokine, IFNgamma, also abolishes suppression of TNFalpha in LPS tolerance. The effect of LPS tolerance on HKSa and IFNgamma-induced inflammatory mediator production is not well defined. We hypothesized that LPS, HKSa and IFNgamma differentially regulate pro-inflammatory mediators and chemokine production in LPS-induced tolerance. THP-1 cells were pretreated for 24 h with LPS (100 ng/ml) or LPS (100 ng/ml) + IFNgamma (1 microg/ml). Cells were subsequently stimulated with LPS or HKSa (10 microg/ml) for 24 h. The production of the cytokines TNFalpha, IL-6, IL-1beta, and GMCSF and the chemokine IL-8 were measured in supernatants. LPS and HKSa stimulated TNFalpha (3070 +/- 711 pg/ml and 217 +/- 9 pg/ml, respectively) and IL-6 (237 +/- 8.9 pg/ml and 56.2 +/- 2.9 pg/ml, p < 0.05, n = 3, respectively) in control cells compared to basal levels (< 25 pg/ml). LPS induced tolerance to secondary LPS stimulation as evidenced by a 90% (p < 0.05, n = 3) reduction in TNFalpha. However, LPS pretreatment induced priming to HKSa as demonstrated by increased TNFalpha (2.7 fold, from 217 to 580 pg/ml, p < 0.05, n = 3 ). In contrast to suppressed TNFalpha, IL-6 production was augmented to secondary LPS stimulation (9 fold, from 237 to 2076 pg/ml, p < 0.01, n = 3) and also primed to HKSa stimulation (62 fold, from 56 to 3470 pg/ml, p < 0.01, n = 3). LPS induced IL-8 production and to a lesser extent IL-1beta and GMCSF. LPS pretreatment did not affect secondary LPS stimulated IL-8 or IL-1beta, although HKSa stimulation augmented both mediators. In addition, IFNgamma pretreatment reversed LPS tolerance as evidenced by increased TNFalpha levels while IL-6, IL-1beta, and GMCSF levels were further augmented. However, IL-8 production was not affected by IFNgamma. These data support our hypothesis of differential regulation of cytokines and chemokines in gram-negative- and gram-positive-induced inflammatory events. Such changes may have implications in the pathogenesis of polymicrobial sepsis.     

Soluble tumour necrosis factor-alpha receptor I and interleukin-6 as markers of activity in thyrotoxic Graves' disease.

Autoimmune thyroid diseases are thought to be mediated by pro-inflammatory cytokines such as TNFalpha and IL-6. Serum levels of cytokines may indicate activity levels of immune functions. We investigated serum levels of IL-6 and of the soluble receptor of TNFalpha in patients with newly diagnosed onset of Graves' hyperthyroidism. The predominantly female group consisted of 39 patients, mean fT4 was 47.6 pg/ml (normal values 7.5=19.0 pg/ml). After diagnosis, all patients were treated with anti-thyroid drugs. Soluble Tumour Necrosis Factor Receptor I (TNF-RI) serum levels were found significantly increased (mean 3.7+/-1.3 ng/ml; p<0,01) compared to a matched group of apparent healthy individuals (mean sTNF-RI 1.8+/-0.5 ng/ml) and to a matched group of patients with treated Graves' disease (mean sTNF-RI 1.9+/-0.6 ng/ml). When IL-6 was assessed only 4 of the 39 patients exhibited increased serum levels. Our finding may indicate that sTNF-RI and possibly its ligand, TNFalpha, could play an important role in the onset of the acute stage of Graves' disease.     

Quantitation of arachidonic acid metabolites in small tissue biopsies by reversed-phase high-performance liquid chromatography

Arachidonic acid metabolites exert a variety of distinct biological effects on the initiation and resolution of inflammatory diseases and their measurements in tissue can be critical to evaluate their regulatory function during the course of inflammation and to supplement in vitro experiments. The aim of this study was the detection and quantitative analysis of four arachidonic acid metabolites in small-sized biopsies of human periodontal tissues. The biopsies were homogenized and injected directly into a single analytical column of a RP-HPLC system. Detection was performed by a photodiode array detector. Calibration was established by dilutions of authentic standards of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), and 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). A total of 38 specimens weighing between 19 and 191 mg (wet tissue) were analyzed (mean = 59.9 +/- 30.2 mg). The detection limits were 1 pg for LTB4 and 12-HETE, 0.5 pg for 15-HETE, and 10 ng for PGE2. The concentrations of PGE2 and LTB4 were significantly higher in inflamed than in healthy periodontal tissues (P = 0.0079; P = 0. 0114). 12-HETE was detected in one biopsy (30 pg/g); 15-HETE was not detected. This method of homogenization, extraction, and analysis of arachidonic acid metabolites by RP-HPLC appears to be well suited for studies of human oral biopsies. Only small tissue samples and minimal laboratory equipment were required for a sensitive analysis.     

Murine pregnancies predisposed to spontaneous resorption show alterations in the concentrations of leukotriene B4 and prostaglandin E2.

Female CBA/J mice mated with DBA/2 males exhibit an increased spontaneous resorption rate (30-35%) in their first pregnancy. Second pregnancies show a decreased resorption rate (15-20%). In contrast, resorption in CBA/J females mated with BALB/c males (identical to DBA/2 at the H-2 major histocompatibility locus) occurs with a frequency of 5-10%. Resorption is preceded by fetoplacental infiltration of natural killer (NK)-like cells and a deficiency in a lipophilic NK-suppressive activity. The eicosanoids leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) are known to modulate NK activity in vitro. We measured the concentrations of LTB4 and PGE2 in extracts of individual fetoplacental units at Day 8 of gestation from (1) primigravid CBA/J x DBA/2 resorption-prone matings (RES); (2) second CBA/J x DBA/2 matings (SEC); and (3) primigravid CBA/J x BALB/c control matings (CON). We detected a significant decrease in the mean concentration of LTB4 in RES fetoplacental units (176.4 +/- 11.8 pg/ml; n = 42) compared with CON and SEC fetoplacental units (570.2 +/- 45.5 pg/ml; n = 21 and 420.2 +/- 59.5 pg/ml; n = 39, respectively). To confirm that the LTB4 deficiency is associated with decreased NK suppression in RES matings, we supplemented RES extracts, in vitro, with exogenous LTB4 (0-500 pg/ml). The effect of the addition of LTB4 to RES extracts was biphasic. Addition of LTB4 in the range of 30-125 pg/ml increased the extract's NK suppressive capacity, whereas LTB4 alone either stimulated NK activity or was without effect. These results suggest a critical role for LTB4 in averting NK-mediated early spontaneous fetal resorption.     

Nicotinic acetylcholine receptor subunits and receptor activity in the epithelial cell line HT29

In the present study we have used RT-PCR to investigate nicotinic acetylcholine receptor (nAChR) subunit expression, and studied the effect of nicotine on TNFalpha-induced cytokine (IL-8) release in the epithelial cell line HT29. RNA was extracted using a commercial kit and amplified by RT-PCR. RT-PCR products were separated by electrophoresis and visualised using ethidium bromide. IL-8 release was measured by ELISA from cells activated for 6 h with TNFalpha (50 ng ml(-1)) in the absence and presence of nicotine (10(-11)-10(-6) M). HT29 cells contained mRNA for beta1, alpha4, alpha5, and alpha7 nAChR subunits. Activation of HT29 cells increased IL-8 release from undetectable amounts to 3.92 +/- 0.51 ng ml(-1) (n = 5). Nicotine significantly inhibited TNFalpha-induced IL-8 release in a concentration related manner with peak inhibition occurring at 10(-7) M (2.39 +/- 0.78 ng ml(-1), n = 5). Our data suggests that, while HT29 cells express mRNA for nAChR subunits, the only nAChR subunits that could form functional receptors and inhibit IL-8 release are alpha7.     

Enhanced superoxide anion generation but reduced leukotriene B4 productivity in thioglycollate-elicited peritoneal macrophages

Lipoxygenase metabolism of arachidonic acid was compared between peritoneal macrophages from untreated rats and those from rats on day 7 after intraperitoneal injection of thioglycollate broth (TG). Resident macrophages (M phi) from untreated rats produced mainly LTB4 (303 +/- 25 pmol/5 x 10(6) cells) and 5-HETE (431 +/- 56 pmol/5 x 10(6) cells) when stimulated with 5 micrograms/ml calcium ionophore A23187 for 20 min at 37 degrees C. On the other hand, TG-elicited M phi generated less amounts of lipoxygenase metabolites (157 +/- 10 pmol LTB4 and 319 +/- 19 pmol 5-HETE/5 x 10(6) cells) with the same stimulus. Then, leukotriene productivity was examined by using subcellular fractions of each M phi lysate and an unstable epoxide intermediate, leukotriene A4. LTA4 hydrolase activity was mainly contained in soluble fractions from the both groups of M phi. The cytosol fraction from the resident M phi exhibited the following specific and total activity; 2.2 +/- 0.1 nmol LTB4/mg protein/5 min and 12.2 +/- 0.5 nmol LTB4/5 min per 10(8) cells. On the contrary, the cytosol fraction from the TG-elicited M phi showed 1.9 +/- 0.1 nmol LTB4/mg protein/5 min and 9.6 +/- 0.3 nmol LTB4/5 min per 10(8) cells. The resident M phi, however, generated 0.14 +/- 0.04 nmol O2-/min/4 x 10(5) cells whereas the TG-elicited M phi did 0.49 +/- 0.13 nmol O2-/min/4 x 10(5) cells when stimulated with wheat germ lectin. These results suggest that the TG-elicited macrophages show enhanced superoxide production but generate less lipoxygenase metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gas chromatographic-mass spectrometric analysis of lipoxygenase products in post-ischemic rabbit myocardium

Leukotriene B4 (LTB4) is a potent chemotactic compound for neutrophils and is thought to be an important mediator of myocardial ischemia-reflow injury. We have measured LTB4 in rabbit cardiac tissue following ischemia-reflow using a sensitive and specific gas chromatographic-mass spectrometric (GC-MS) assay. The concentration of LTB4 in rabbit myocardium following 45 min ischemia and 3 h reflow was 48.7 +/- 12.5 pg/g, significantly higher than in non-ischemic tissue from the same animal (17.5 +/- 3.9 pg/g). These concentrations were at least an order of magnitude lower than previously reported values assessed by radioimmunoassay (RIA). Compared with the GC-MS method, RIA greatly overestimated LTB4 concentrations in cardiac tissue. The capacity of post-ischemic myocardium to produce lipoxygenase products, LTB4, 5-, 12- and 15-HETEs was also assessed following incubation of myocardium ex vivo with calcium ionophore. In all animals ischemic cardiac tissue produced greater amounts of LTB4, 5-, and 12-HETEs than non-ischemic myocardium and 12-HETE was the major product. Neutrophils that have accumulated in the injured tissue may be a major source of these products. However, in contrast to cardiac tissue, isolated rabbit neutrophils stimulated with A23187 produced 5-HETE as the major product with very little 12-HETE formed. These latter findings suggest that cells other than neutrophils may contribute to the production of lipoxygenase products during myocardial ischemia-reflow injury.     

Characterization of leukotriene B4 synthesis in canine polymorphonuclear leukocytes.

The synthesis and release of leukotriene B4 (LTB4) from canine polymorphonuclear leukocytes (PMNs) was characterized in terms of incubation time, temperature and effects of calcium ionophore A23187 concentrations. Maximal LTB4 concentrations were determined when canine PMNs were incubated with 10 microM A23187. Increasing LTB4 concentrations were determined through 10 min incubation. The maximal LTB4 concentrations (310 +/- 30 pg LTB4/2.5 x 10(5) cells) determined at 10 min did not change through a 55 min incubation period. Greater LTB4 concentrations were synthesized by canine PMNs at 37 degrees C (268 +/- 12 pg LTB4/2.5 x 10(5) cells) than at 25 degrees C (206 +/- 11 pg LTB4/2.5 x 10(5) cells) or 5 degrees C (59 +/- 3 pg LTB4/2.5 x 10(5) cells). The synthesis of LTB4 in canine PMNs was inhibited by incubation of the cells with either of two known lipoxygenase inhibitors, BWA4C or BW755C. BWA4C inhibited LTB4 synthesis with an approximate IC50 = 0.1 microM, whereas BW755C inhibited LTB4 synthesis with an approximate IC50 = 10 microM. These results indicate canine PMNs have the capability to synthesize large quantities of LTB4 when stimulated with calcium ionophore A23187. Furthermore, the 5-lipoxygenase inhibitors BWA4C, an acetohydroxyamic acid, and BW755C, a phenyl pyrazoline, can readily inhibit LTB4 synthesis in canine PMNs.     

Enhanced 5-lipoxygenase activity in lung macrophages compared to monocytes from normal subjects

We compared lipoxygenase activities of lung macrophages obtained from bronchoalveolar lavage to activities of blood monocytes purified by using discontinuous plasma/Percoll density gradients and adherence to tissue culture plastic in five normal subjects. Cells were incubated with ionophore A23187 (10(-9) to 10(-5) M) or arachidonic acid (0.12 to 80 microM) for 1 to 60 min at 37 degrees C to construct dose-response and time-dependence curves of lipoxygenase product generation. Products were identified and were quantified by using high-pressure liquid chromatography and ultraviolet spectroscopy. Under all conditions of product generation, both macrophages and monocytes generated predominantly (5S,12R)-dihydroxy-(6Z, 8E, 10E, 14Z)-eicosatetraenoic acid (leukotriene B4 (LTB4] and (5S)-hydroxy-(6E, 8Z, 11Z, 14Z) - eicosatetraenoic acid (5 - HETE), but, in each subject, macrophages invariably released greater amounts of LTB4 and 5-HETE than monocytes. In response to A23187, macrophages released a maximum of 183 +/- 96 pmol of LTB4 and 168 +/- 108 pmol of 5-HETE per 10(6) cells (mean +/- SEM), whereas monocytes released only 16 +/- 1 and 18 +/- 8 pmol per 10(6) cells of LTB4 and 5-HETE, respectively. After adding arachidonic acid, macrophages released a maximum of 52 +/- 21 pmol of LTB4 and 223 +/- 66 pmol of 5-HETE, whereas monocytes released no detectable products. The results suggest that mononuclear phagocyte maturation in the lung may be accompanied by an enhanced ability to generate 5-lipoxygenase products.     

The differences in 12-hydroxyeicosatetraenoic acid and thromboxane B2 release from guinea pig platelets.

Anti-12(S)-hydroxyeicosatetraenoic acid (12-HETE)-antibody and anti-thromboxane B2 (TXB2)-antibody were generated and applied to the radioimmunoassay. The detection limit for 12-HETE was 16 pg. The cross-reactivities of anti-12-HETE-antibody were 4.6% for 15-HETE, 0.18% for 5-HETE and below 0.15% for leukotrienes and prostaglandins (PGs). 12-HETE and TXB2 released from guinea pig platelets were measured by radioimmunoassay. Platelet activating factor (PAF) at 10(-9) M induced the aggregation of platelets, the releases of immunoreactive-12-HETE (1.8 +/- 1.2 ng/10(8) platelets, mean +/- S.D.) and immunoreactive-TXB2 (18.5 +/- 17.3 ng/10(8) platelets). Collagen at 1 microgram/ml also evoked platelet aggregation, the releases of immunoreactive-12-HETE (2.7 +/- 1.1 ng/10(8) platelets) and immunoreactive-TXB2 (11.8 +/- 4.6 ng/10(8) platelets). By the stimulation with these compounds, TXB2 was produced in a greater amount than 12-HETE from guinea pig platelets. Although 10(-7) M and 10(-6) M U46619, a TXA2 mimetic, caused platelet aggregation, arachidonic acid metabolites were not released. These data suggest the presence of different mechanisms of platelet activation depending on each stimulus.     

Transplantation of amniotic membrane in corneal ulcers and persistant epithelial defects

Amniotic membrane transplantation (AMT) leads to reduction of inflammatory symptoms and causes faster epithelisation in corneal ulcers and persistant epithelial defect. 21 patients with corneal ulcer (n = 18) or non-healing epithelial defect (n = 3) unresponsive to conventional treatment were included in the study. All patients were treated by AMT. Corneal epithelial cells in patients suffering from corneal ulcer secreted 3.51 +/- 1.79 of IL-1alpha, 64.27 +/- 31.53 pg/mL of TNFalpha and 209.07 +/- 201.82 pg/mL of VEGF. Levels of all 3 investigated cytokines were significantly higher as compared to controls (p < 0.005). Amniotic membranes that were used contained 775.69 +/- 613.98 pg/mL of IL-1alpha, 0.036 +/- 0.033 pg/mL of sTNF and 175.01 +/- 166.63 pg/mL of VEGF-R. Supporting effect of the AMT could be explained by the fact that AM secretes its natural antinflammatory antagonists IL-1ra, sTNF and VEGF-R.     

15-Hydroxy-eicosatetraenoic acid (15-HETE) inhibits carrageenan-induced experimental arthritis and reduces synovial fluid leukotriene B4 (LTB4)

15-Hydroxy-eicosatetraenoic acid (15-HETE), a product of arachidonic acid, has no proinflammatory capacity, but can inhibit the formation and the chemotactic response of neutrophils to leukotriene B4 (LTB4), a potent mediator of inflammation. The purpose of the present study was to determine whether intraarticular administration of 15-HETE in carrageenan-induced acute arthritis might decrease the levels of LTB4 in synovial fluid and modify the arthritis. A bilateral acute knee joint arthritis was established in 7 dogs by intraarticular injections of carrageenan every third day. To the right joints, 15-HETE was administered both concomitantly with the carrageenan injections and continuously via an osmotic pump. In samples of synovial fluid obtained on day 0, 3 and 10 PGE2 and LTB4 were determined using reversed phase high performance liquid chromatography combined with radioimmunoassays and neutrophil chemokinesis. In the presence of 15-HETE the clinical severity of arthritis was significantly reduced and the volume of synovial effusate was decreased on an average by 42%. Furthermore, the relative number of neutrophils in histological sections of synovial tissue was decreased by 58%. Intraarticular caragheenan injections induced LTB4 formation, and maximum levels were obtained on day 3 (279.2 +/- 148.2 pg/joint). PGE2 was also present on day 3, but maximum levels were detected on day 10 (9.5 +/- 4.8 ng/joint). In joints injected with both carragheenan and 15-HETE the levels of LTB4 on days 3 and 10 were inhibited by 90% and 83%, respectively. For PGE2 a small but insignificant decrease was found on both day 3 and on day 10. These results show that LTB4 may be an important mediator of acute arthritis induced by carragheenan in dogs, and that intraarticular administration of 15-HETE can modify this arthritis by inhibiting LTB4 formation.     

The negative immunoregulatory effects of serotonin (5-HT) moduline,an endogenous 5-HT1B receptor antagonist with anti-anxiety properties

BACKGROUND: Serotonin (5-HT) has negative immunoregulatory effects by reducing the interferon-gamma (IFNgamma)/interleukin-10 (IL-10) production ratio by stimulated immune cells. Leukocytes have functional 5-HT1B receptors. 5-HT moduline, an endogenous 5-HT1B receptor antagonist, may antagonize the 5-HT1B agonist-induced proliferation of immune cells. AIMS: To examine the effects of 5-HT moduline on the stimulated production of IFNgamma, tumor necrosis factor alpha (TNFalpha) and IL-10. RESULTS: 5-HT moduline, 10(-6) M and 10(-5)M, significantly reduced the production of IFNgamma and the IFNgamma/IL-10 ratio. 5-HT moduline 10(-5)M significantly reduced the production of TNFalpha. The combination of 5-HT, 15 microg/mL, with 5-HT moduline, 10(-6)M and 10(-5)M, further decreases the IFNgamma/IL-10 production ratio. INTERPRETATION: 5-HT moduline has negative immunoregulatory effects.     

In humans, corticotropin releasing hormone antagonizes some of the negative immunoregulatory effects of serotonin

Both corticotropin releasing hormone (CRH) and serotonin (5-HT) participate in the stress response and are known to modulate cytokine release by human immune cells. Extracellular 5-HT concentrations at or above the serum values have negative immunoregulatory effects by inhibiting the production of interferon-gamma (IFNgamma), a pro-inflammatory cytokine produced by Th-1-like lymphocytes, whereas 5-HT has no significant effects on the production of interleukin-10 (IL-10), an anti-inflammatory cytokine. In one study, CRH significantly decreases IFNgamma production by cultured human peripheral blood immunocytes, whereas in other studies CRH increases the production of cytokines, such as IL-1, IL-2 and IL-6. The aims of the present study were to examine i) the effects of CRH, 10-9 M, 10(-8) M and 10(-7) M, on the stimulated production of IFNgamma, IL-10 and tumor necrosis factor-alpha (TNFalpha) by human whole blood; and ii) whether CRH, 10(-9) M, 10(-8) M and 10(-7) M, may antagonize some of the negative immunoregulatory effects of 5-HT, 1.5 microg/mL or 15 microg/mL. We found that CRH, 10(-9) M, 10(-8) M and 10(-7) M, had no significant effects either on the stimulated production of IFNgamma, IL-10 or TNFalpha or on the IFNg/IL-10 production ratio, which reflects the pro-inflammatory capacity of the culture. 5-HT, 1,5 microg/dL and 15 microg/dL, significantly suppressed the production of IFNgamma and TNFalpha and the IFNg/IL-10 production ratio. CRH, 10(-7) M, significantly reversed the 5-HT (1.5 microg/mL and 15 microg/mL)-induced suppression of IFNg production. CRH at all concentrations significantly blocked the 5-HT (1.5 microg/mL and 15 microg/mL)-induced suppression of TNFalpha production. The results suggest that CRH has no significant direct effects on the production of IFNgamma, IL-10 and TNFalpha, but antagonizes the negative immunoregulatory effects of 5-HT on the production of IFNgamma and TNFalpha and on the IFNgamma/IL-10 production ratio.     

Creatine supplementation reduces plasma levels of pro-inflammatory cytokines and PGE<Subscript>2</Subscript> after a half-ironman competition

OBJECTIVE: The effect of creatine supplementation upon plasma levels of pro-inflammatory cytokines: Interleukin (IL) 1 beta and IL-6, Tumor Necrosis Factor alpha (TNFalpha), and Interferon alpha (INF alpha) and Prostaglandin E(2) (PGE(2)) after a half-ironman competition were investigated. METHODS: Eleven triathletes, each with at least three years experience of participation in this sport were randomly divided between the control and experimental groups. During 5 days prior to competition, the control group (n = 6) was supplemented with carbohydrate (20 g x d(-1)) whereas the experimental group (n = 5) received creatine (20 g x d(-1)) in a double-blind trial. Blood samples were collected 48 h before and 24 and 48 h after competition and were used for the measurement of cytokines and PGE(2). RESULTS: Forty-eight hours prior to competition there was no difference between groups in the plasma concentrations (pg x ml(-1), mean +/- SEM) of IL-6 (7.08 +/- 0.63), TNFalpha (76.50 +/- 5.60), INF alpha (18.32 +/- 1.20), IL-1 beta (23.42 +/- 5.52), and PGE(2) (39.71 +/- 3.8). Twenty-four and 48 h after competition plasma levels of TNFalpha, INF alpha, IL-1 beta and PGE(2) were significantly increased (P < 0.05) in both groups. However, the increases in these were markedly reduced following creatine supplementation. An increase in plasma IL-6 was observed only after 24 h and, in this case, there was no difference between the two groups. CONCLUSION: Creatine supplementation before a long distance triathlon competition may reduce the inflammatory response induced by this form of strenuous of exercise.     

High altitude increases circulating interleukin-6, interleukin-1 receptor antagonist and C-reactive protein

Hypoxic pulmonary vasoconstriction is associated with but may not be sufficient for the development of high-altitude pulmonary oedema (HAPO). Hypoxia is known to induce an inflammatory response in immune cells and endothelial cells. It has been speculated that hypoxia-induced inflammatory cytokines at high altitude may contribute to the development of HAPO by causing capillary leakage in the lung. We were interested if such an inflammatory response, possibly involved in a later development of HAPO, is detectable at high altitude in individuals without HAPO. We examined the plasma levels of interleukin 6 (IL-6), interleukin 1 receptor antagonist (IL-1ra) and C-reactive protein (CRP) in two independent studies: study A, Jungfraujoch, Switzerland, three overnight stays at 3458 m, n=12; study B: Capanna Regina Margherita, Italy, 3 overnight stays at 3647 m and one overnight stay at 4559 m, n=10. In both studies, probands showed symptoms of acute mountain sickness but no signs of HAPO. At the Jungfraujoch, IL-6 increased from 0.1+/-0.03 pg/ml to 2. 0+/-0.5 pg/ml (day 2, P=0.03), IL-1ra from 101+/-21 to 284+/-73 pg/ml (day 2, P=0.01), and CRP from 1.0+/-0.4 to 5.8+/-1.5 micrograms/ml (day 4, P=0.01). At the Capanna Margherita, IL-6 increased from 0. 5+/-0.2 pg/ml to 2.0+/-0.8 pg/ml (P=0.02), IL-1ra from 118+/-25 to 213+/-28 pg/ml (P=0.02), and CRP from 0.4+/-0.03 to 3.5+/-1.1 micrograms/ml (P=0.03). IL-8 was below the detection limit of the ELISA (<25 pg/ml) in both studies. The increase of IL-6 and IL-1ra in response to high altitude was delayed and preceded the increase of CRP. We conclude that: (1) circulating IL-6, IL-1ra and CRP are upregulated in response to hypobaric hypoxic conditions at high altitude, and (2) the moderate systemic increase of these inflammatory markers may reflect considerable local inflammation. The existence and the kinetics of high altitude-induced cytokines found in this study support the hypothesis that inflammation is involved in the development of HAPO.     

Cell-cell interaction of macrophages and vascular smooth muscle cells in the synthesis of leukotriene b(4)

Biosynthesis of LTB(4) during cell-cell interaction between vascular smooth muscle cells (SMC) and alveolar macrophages (AM) has been investigated by use of both high-pressure Hquid chromatography (HPLC) and radtoimmunoassay (RIA). Both interleukin-beta (IL-beta) and tumour necrosis factor-alpha (TNFalpha) induced a time- and dose-dependent synthesis of 15-, and 5-hydroxyeicosatetraenoic acids (HETEs) from cultured SMC. However, neither TNFalpha nor IL-1beta induced a significant LTB(4) production in SMC alone or AM alone after 24 h of incubation. Addition of IL-1beta and TNFalpha simultaneously to SMC resulted in a dose-dependent synergistic increase of HETEs. Macrophages dose-dependently transformed extremely low concentrations of exogenous LTA(4) into LTB(4). Incubation of vascular SMC with various numbers of AM in the presence of IL-1beta (5 units/ml) and TNFalpha (10 units/ml) induced a great increase of LTB(4) synthesis in comparison with the detectable levels of LTB(4) produced by macrophages alone. Pretreatment of SMC with NDGA, cycloheximide, and actinomycin not only inhibited IL-1 and TNT induced HETEs synthesis but also abolished LTB(4) production when co-incubated with macrophages. These results suggest that LTB(4) in a mixture of SMC and macrophages could originate from a transcellular metabolism, i.e. macrophages transforming SMC-derived LTA(4) into LTB(4).     

Effects of treatment with LH or FSH from 4 to 8 weeks of age on the attainment of puberty in bull calves

A transient increase in gonadotropin secretion between 6 and 20 weeks of age is critical for the onset of puberty in bull calves. To try and hasten the onset of puberty, bull calves were treated (s.c.) with 3 mg of bLH (n = 6) or 4 mg of bFSH (n = 6) once every 2 days, from 4 to 8 weeks after birth; control calves received saline (n = 6). At 4 and 8 weeks of age, mean LH concentrations were higher (P < 0.05) in bLH-treated (2.3 +/- 0.04 ng/ml and 1.20 +/- 0.04 ng/ml) as compared to control calves (0.50 +/- 0.1 ng/ml and 0.70 +/- 0.10 ng/ml). Mean serum FSH concentrations at 4 and 8 weeks of age, were higher (P < 0.05) in bFSH-treated (1.60 +/- 0.20 ng/ml and 1.10 +/- 0.2 ng/ml) as compared to control calves (0.38 +/- 0.07 ng/ml and 0.35 +/- 0.07 ng/ml). The age at which scrotal circumference (SC) first reached > or = 28 cm, occurred earlier (P < 0.05) in bFSH-treated calves as compared to saline-treated calves (39.3 +/- 1.3 and 44.8 +/- 1.3 weeks of age, respectively). Based on testicular histology at 56 weeks of age, treatment with bFSH resulted in greater (P < 0.05) numbers of Sertoli cells (5 +/- 0.2, 6 +/- 0.3 and 5 +/- 0.3 in bLH-, bFSH- and saline-treated calves, respectively); elongated spermatids (42 +/- 2, 57 +/- 8 and 38 +/- 5 in bLH-, bFSH- and saline-treated calves, respectively) and spermatocytes (31 +/- 3, 38 +/- 3 and 29 +/- 2 in bLH-, bFSH- and saline-treated calves, respectively) per seminiferous tubule. We concluded that treatment of bull calves with bFSH from 4 to 8 weeks of age increased testicular growth (SC); hastened onset of puberty (SC > or = 28 cm); and enhanced spermatogenesis.     

Relationship among nitric oxide, leptin, ACTH, corticosterone, and IL-1beta, in the early and late phases of adjuvant arthritis in male Long Evans rats

Leptin, a hormone regulating body weight, food intake, and metabolism, is associated with activation of immune cells and inflammation. In this study we analyzed levels of leptin, adrenocorticotropic hormone (ACTH), corticosterone, interleukin 1beta (IL-1beta), and nitric oxide (NO) production on days 10 and 22 of adjuvant arthritis (AA) in male Long Evans rats to ascertain possible relationship of leptin with its modulators during the early and late phases of chronic inflammation. The circulating leptin levels were significantly reduced already on day 10 of AA compared to controls (1.97+/-0.22 ng/ml vs. 3.08+/-0.25 ng/ml, p<0.05); on day 22 no significant further drop was observed (1.06+/-0.21 ng/ml). Leptin mRNA in epididymal fat tissue was reduced in arthritic animals compared to controls on day 22 (0.61+/-0.09 vs. 1.30+/-0.1 arbU/GAPDH (p<0.01). IL-1beta concentration in spleen was enhanced on day 10 of AA (24.55+/-4.67 pg/100 microg protein vs. 14.33+/-1.71 pg/100 microg protein; p<0.05); on day 22 it did not differ from controls. ACTH and corticosterone levels were significantly elevated only on day 22 of AA (ACTH: 306.17+/-42.22 pg/ml vs. 157.61+/-23.94 pg/ml; p<0.05; corticosterone: 5.24+/-1.38 microg/100 ml vs. 1.05+/-0.23 microg/100 ml; p<0.01). Nitrate levels were enhanced similarly on days 10 (49.86+/-1.83 microM) and 22 of AA (43.58+/-2.17 microM), compared to controls (23.42+/-1.39 microM, p<0.001). These results show that corticosterone does not stimulate leptin production during AA. The suppression of leptin may be a consequence of permanent activation of NO, IL-1beta, and of lower weight gain. Circulating leptin does not seem to play a key role in the progression of chronic arthritis.