PGE2 enhanced TNFα-mediated IL-8 induction in monocytic cell lines and PBMC.

BACKGROUND & PURPOSE: Recent studies suggested a role of prostaglandin E2 (PGE2) in the expression of the chemokine IL-8 by monocytes. The function of EP4 receptor for TNFα-induced IL-8 expression was studied in monocytic cell lines. EXPERIMENTAL APPROACH: IL-8 mRNA and protein induction as well as IL-8 promoter activity and transcription factor activation were assessed in monocytic cell lines, primary blood mononuclear cells (PBMC) and transgenic HEK293 cells expressing the EP4 receptor. KEY RESULTS: In monocytic cell lines THP-1, MonoMac and U937 PGE2 had only a marginal impact on IL-8 induction but strongly enhanced TNFα-induced IL-8 mRNA and protein synthesis. Similarly, in PBMC IL-8 mRNA induction was larger by simultaneous stimulation with TNFα and PGE2 than by either stimulus alone. The EP4 receptor subtype was the most abundant EP receptor in all three cell lines and in PBMC. Stimulation of THP-1 cells with an EP4 specific agonist enhanced TNFα-induced IL-8 mRNA and protein formation to the same extent as PGE2. In HEK293 cells expressing EP4, but not in wild type HEK293 cells lacking EP4, PGE2 enhanced TNFα-induced IL-8 protein and mRNA synthesis. In THP-1 cells, the enhancement of TNFα-mediated IL-8 mRNA induction by PGE2 was mimicked by a PKA-activator. Furthermore in these cells PGE2 induced expression of transcription factor C/EBPß, enhanced NF-κB activation by TNFα and inhibited TNFα-mediated AP-1 activation. PGE2 and TNFα synergistically activated transcription factor CREB, induced C/EBPß expression and enhanced the activity of an IL-8 promoter fragment containing -223 bp upstream of the transcription start site. CONCLUSIONS AND IMPLICATIONS: These findings suggest that a combined stimulation of TNFα and PGE2/EP4 signal chains in monocytic cells leads to maximal IL-8 promoter activity, as well as IL-8 mRNA and protein induction, by activating the PKA/CREB/C/EBPß as well as NF-κB signal chains.

Reference range of gestational uterine artery resistance index in small canine breeds.

The objectives of this study were to evaluate the uterine artery blood flow during the second half of normal pregnancy in small breed dogs and to determine the RI reference values of this artery in these bitches. Fifty-two, 1.5-6.5 kg, purebred healthy pregnant bitches were included in this study. Doppler ultrasonographic examinations of the uterus were performed every 10 days from Day 30 of the estrous cycle to parturition. Peak systolic velocity (PSV) and end diastolic velocity (EDV) of the uterine artery were measured while RI [(PSV-EDV)/PSV] was automatically calculated. Values of PSV, EDV and RI were analyzed by repeated measures ANOVA followed by Tukey test using age, body weight, litter size and parity as covariates. Resistance index reference ranges were constructed by regressing this index on gestational age. Equation of the polynomial regression curves was used to calculate the mean, 95th and 5th centiles for each gestational age (GA). A gradual increase of PSV (P < 0.01) and EDV (P < 0.01) and a decrease in RI (P < 0.01) were found throughout the study period. PSV and EDV were influenced by litter size (P < 0.05), while RI was not. None of the Doppler parameters were influenced by age, body weight or parity. A second-degree polynomial described the relationship between RI of uterine artery and GA in the different time points: [RI = 0.993 + 8.4x10-5 x GA2- 0.0117 x GA]. It is concluded that uterine artery blood flow progressively increased throughout normal pregnancy in small breed bitches. In addition, reference ranges of uterine artery RI were 0.64-0.79, 0.57 to 0.75, 0.53 to 0.71 and 0.52 to 0.66 on Days 30, 40, 50 and 60 of gestation, respectively.

Evaluation of feline uterine and umbilical arteries blood flow in a pharmacologically induced abnormal gestation model.

The aim of this study was to describe resistance index (RI) and systolic/diastolic ratio (S/D ratio) of uterine and umbilical arteries in an experimental model of abnormal pregnancy in felids. On days 30 to 35 (32 ± 2.9) after mating, 20 domestic short-hair pregnant queens were randomly assigned to one of the following treatment groups: a treated group (TG; n = 8), which received 10 mg/kg of aglepristone subcutaneously twice, 24 hours apart, and a control nontreated group (CG; n = 12). M-mode and Doppler ultrasonographic evaluations were performed at the initiation of the treatment (Day 0) and then every other day during 8 days. In both groups, uterine and umbilical arteries were evaluated by Doppler ultrasound, whereas fetal heart rate was assessed by M-mode ultrasound. Resistance index of uterine artery augmented in TG from Day 2 onward, conversely it decreased in CG (P < 0.01). On Day 8, RI values were 0.64 ± 0.05 vs 0.37 ± 0.01 for TG and CG, respectively. Additionally, S/D ratio of the same artery presented an increase in TG, whereas this ratio diminished in CG (P < 0.01). On Day 8, this parameter showed values of 2.98 ± 0.4 vs 1.62 ± 0.06 for TG and CG, respectively. Resistance index of umbilical artery remained almost unchanged in TG from Day 6 onward, whereas it progressively decreased in CG throughout the course of the study (P < 0.05). On Day 8, RI were 0.89 ± 0.04 and 0.82 ± 0.01, for TG and CG, respectively. Furthermore, on Day 8, S/D ratio of umbilical artery progressively diminished in CG but not in TG (P < 0.01), being 14.7 ± 9.1 vs 5.9 ± 0.3 for TG and CG, respectively. Fetal heart rate was higher in TG than in CG (P < 0.05). Group differences in Doppler parameters appeared on Day 2, when the other clinical or ultrasonographic signs were still absent. It is concluded that blood flow of the uterine and umbilical arteries differed between these normal and abnormal gestations predicting an adverse obstetric outcome.

Uterine blood flow evaluation in bitches suffering from cystic endometrial hyperplasia (CEH) and CEH-pyometra complex.

Doppler ultrasound is a useful diagnosis tool to evaluate uterine blood flow in different canine reproductive states. The aim of this study was to describe and compare uterine blood flow in bitches suffering from cystic endometrial hyperplasia (CEH) and CEH-pyometra complex (CEH-P). Ninety diestrous bitches were clinically, hematologically, and ultrasonographically classified into four groups: (1) clinical signs, leukocytosis, CEH, and uterine luminal contents (CEH-P, n = 31); (2) asymptomatic bitches with CEH and uterine contents (CEH-C, n = 15); (3) asymptomatic bitches with CEH without uterine contents (CEH, n = 16); and (4) normal diestrous bitches (ND, n = 28). The widest cross-sectional diameter and uterine wall thickness (W) of uterine horns were measured using two-dimensional ultrasound. Peak systolic velocity and end diastolic velocity of uterine arteries were measured by Doppler ultrasound in all the bitches. Peak systolic velocity and end diastolic velocity were higher in CEH-P than in CEH-C, CEH, and ND (P < 0.01); although both parameters did not show differences among the latter three groups (P > 0.1). Conversely, resistance index (RI) reported lower values in CEH-P than that in the other three groups (P < 0.01), being ND higher (P < 0.01) than both CEH-C and CEH which did not differ between themselves (P > 0.1). When all the bitches were considered, correlation between RI and cross-sectional diameter was r = -0.69 (P < 0.01) and between RI and uterine wall thickness was r = -0.02 (P > 0.1). It is concluded that the uterine artery blood flow velocity of bitches suffering from pyometra was higher, not only from normal bitches, but also from females with endometrial hyperplasia. Furthermore, endometrial hyperplasia, accompanied or not by luminal contents, had a higher blood flow velocity than normal uterus. Hemodynamic parameters appear as useful markers to differentiate uterine pathologic conditions.

Doppler ultrasonographic assessment of maternal and fetal arteries during normal feline gestation.

The aim of this study was to describe Doppler parameters of uterine, umbilical, fetal abdominal aorta, fetal renal and fetal internal carotid arteries, as well as fetal heart rate (FHR), during normal feline gestation. Fifteen, 1-4 years of age, weighing 2.5-3.9kg, domestic short-hair pregnant queens, which were born in our institutional cat colony were included in this study. Color and pulsed-wave Doppler evaluations of uterine arteries were performed every 10 days (Day 0, 10, 20, 30, 40, 50, 60) from mating. Fetal Doppler and M-mode ultrasonography were performed to assess umbilical, fetal abdominal aorta, fetal renal, fetal internal carotid arteries and FHR. Both peak systolic velocity (PSV) and end diastolic velocity (EDV) of uterine artery increased up to parturition (P<0.01), while resistance index (RI) decreased from Day 10 onwards (P<0.01). From Day 40 onwards, RI of umbilical artery diminished, while PSV and EDV augmented (P<0.01). Fetal abdominal aorta (P<0.01), renal (P<0.01) and internal carotid (P<0.01) arteries diminished their RI from Days 40, 60 and 40 onwards, respectively. Both PSV and EDV of these three arteries increased progressively. Fetal heart rate was first registered on Day 20 when it began to increase up to Day 40 and then diminished to the end of gestation (P<0.01). It is concluded that blood flow of uterine, umbilical, fetal abdominal aorta, fetal renal and fetal internal carotid arteries progressively increased during normal feline pregnancy, while FHR rose to mid gestation and then decreased up to parturition.

NF-κB-dependent IL-8 induction by prostaglandin E(2) receptors EP(1) and EP(4).

BACKGROUND AND PURPOSE: Recent studies suggested a role for PGE(2) in the expression of the chemokine IL-8. PGE(2) signals via four different GPCRs, EP(1) -EP(4) . The role of EP(1) and EP(4) receptors for IL-8 induction was studied in HEK293 cells, overexpressing EP(1) (HEK-EP(1) ), EP(4) (HEK-EP(4) ) or both receptors (HEK-EP(1) + EP(4) ). EXPERIMENTAL APPROACH: IL-8 mRNA and protein induction and IL-8 promoter and NF-κB activation were assessed in EP expressing HEK cells. KEY RESULTS: In HEK-EP(1) and HEK-EP(1) + EP(4) but not HEK or HEK-EP(4) cells, PGE(2) activated the IL-8 promoter and induced IL-8 mRNA and protein synthesis. Stimulation of HEK-EP(1) + EP(4) cells with an EP(1) -specific agonist activated IL-8 promoter and induced IL-8 mRNA and protein, whereas a specific EP(4) agonist neither activated the IL-8 promoter nor induced IL-8 mRNA and protein synthesis. Simultaneous stimulation of HEK- EP(1) + EP(4) cells with both agonists activated IL-8 promoter and induced IL-8 mRNA to the same extent as PGE(2) . In HEK-EP(1) + EP(4) cells, PGE(2) -mediated IL-8 promoter activation and IL-8 mRNA induction were blunted by inhibition of IκB kinase. PGE(2) activated NF-κB in HEK-EP(1) , HEK-EP(4) and HEK-EP(1) + EP(4) cells. In HEK-EP(1) + EP(4) cells, simultaneous activation of both receptors was needed for maximal PGE(2) -induced NF-κB activation. PGE(2) -stimulated NF-κB activation by EP(1) was blocked by inhibitors of PLC, calcium-signalling and Src-kinase, whereas that induced by EP(4) was only blunted by Src-kinase inhibition. CONCLUSIONS AND IMPLICATIONS: These findings suggest that PGE(2) -mediated NF-κB activation by simultaneous stimulation of EP(1) and EP(4) receptors induces maximal IL-8 promoter activation and IL-8 mRNA and protein induction.

Doppler ultrasonographic assessment of maternal and fetal blood flow in abnormal canine pregnancy.

The aim of this study was to describe the changes of uterine artery, umbilical artery and fetal abdominal aorta, renal and internal carotid arteries blood flow in abnormal canine pregnancy. Twenty-two, Brucella-negative pregnant bitches were retrospectively classified into abnormal (which had either interrupted their pregnancy between days 52 and 60 or had perinatal death >60% of the litter; n=11) and normal (which had delivered healthy puppies at term; n=11). In all the animals, color and pulsed-wave Doppler examinations of uterine artery were conducted every 10 days from Day 20 to 50 from estimated luteinizing hormone peak. Doppler ultrasonography was also conducted in the fetuses to assess umbilical artery, abdominal aorta, renal and internal carotid arteries from Day 40 to 60 of gestation. Throughout the study, resistance index (RI) of uterine, umbilical and fetal renal arteries decreased up to -15% compared to -36% (P<0.01), -11% compared to -23% (P<0.05) and 2% compared to -13% (P<0.05), respectively in the abnormal and normal bitches. Fetal abdominal aorta and internal carotid did not differ between groups (P>0.05). It is concluded that in dogs, uterine artery, umbilical artery and fetal renal artery RI differ between normal and abnormal gestation being useful for the prediction of adverse obstetric outcome.

An experimental model to study resistance index and systolic/diastolic ratio of uterine arteries in adverse canine pregnancy outcome.

The aim of this study was to describe the changes in the resistance index (RI) and systolic/diastolic ratio (S/D) of the uterine arteries during mid-pregnancy abortion induction in the dog. Sixteen 30-35 day pregnant bitches were randomly assigned to either a pharmacological protocol to interrupt gestation (n = 8) or were used as untreated control group (n = 8). Doppler assessments of uterine arteries blood flow were carried out before the initiation of the protocol and then every other day up to abortion (treated group) or parturition (control group). All treated bitches aborted 6 +/- 1.2 days after initiation of the treatment (while none of the non-treated bitches aborted). Pre-treatment RI and S/D did not differ between groups (p > 0.2) while average post-treatment indexes were (mean +/- SD): 0.62 +/- 0.1 vs 0.53 +/- 0.1 (p < 0.01) and 2.96 +/- 0.9 vs 2.23 +/- 0.3 (p = 0.01), for the treated and non-treated group respectively. Correlations between days to abortion and RI or S/D were 0.75 (p < 0.01) and 0.79 (p < 0.01) and, -0.78 (p < 0.01) and -0.73 (p < 0.01) for the treated and non-treated groups respectively. In the treated group, correlations between serum progesterone (P(4)) concentrations and RI and S/D were -0.76 (p < 0.01) and -0.59 (p < 0.01) respectively. It is concluded that, during induction of abortion, RI and S/D of uterine arteries progressively increased while P(4) decreased.