Suppression of fever at near term is associated with reduced COX-2 protein expression in rat hypothalamus

The fever response is blunted at near term. As the enzyme cyclooxygenase-2 (COX-2) plays a critical role in fever development, we measured its expression in rat hypothalamus during pregnancy and lactation. Western blot analysis revealed a 72-kDa COX-2-immunoreactive band in non-immune-challenged, pregnant rats at day 15 of pregnancy. In contrast, it was almost undetectable at near term and at lactation day 5. COX-2 was significantly induced at the 15th day of pregnancy and at the 5th lactating day after intraperitoneal lipopolysaccharide (50 microg/kg). However, this COX-2 induction was significantly reduced at near term compared with values before and after term. The protein levels of the EP3 receptor in the hypothalamus, one of the prostaglandin E(2) (PGE(2)) receptors suggested to be a key receptor for fever induction, were unaffected throughout the pregnancy and lactation in both non-immune-challenged and lipopolysaccharide-treated rats. These data suggest that suppression of fever at near term is associated with a significantly reduced induction of COX-2 by lipopolysaccharide, resulting in a reduced production of PGE(2). Altered expression of the EP3 receptor does not seem to be involved in this fever refractoriness at near term.     

Interleukin-1 receptor antagonist as a modulator of gender differences in the febrile response to lipopolysaccharide in rats

Febrile responses to bacterial pathogens are attenuated near term of pregnancy in several mammalian species. It is unknown, however, whether this reflects a fundamental physiological adaptation of female rats or whether it is specific to pregnancy. The aims of this study therefore were 1) to determine whether febrile responses to the bacterial endotoxin lipopolysaccharide (LPS) are attenuated in female vs. male rats and, if so, to identify possible mechanisms involved in modulating this and 2) to assess whether plasma concentrations of the anti-inflammatory cytokine, interleukin-1 receptor antagonist (IL-1ra), an important regulator of fever, are dependent on the physiological state of the female and could therefore be involved in modulating febrile responses. We found febrile responses were attenuated in cycling female vs. male rats and also in near-term pregnant dams vs. cycling females after intraperitoneal injection of LPS (0.05 mg/kg). Plasma levels of IL-1ra were significantly greater in female rats after injection of LPS, particularly during pregnancy, than in males. This was accompanied by attenuated levels of hypothalamic IL-1beta and cyclooxygenase-2 mRNA, two key mediators of the febrile response, in female rats. Furthermore, increasing plasma levels of IL-1ra in male rats by intraperitoneal administration of the recombinant antagonist attenuated hypothalamic mRNA levels of these mediators after LPS. These data suggest that there is a fundamental difference in febrile response to LPS between the genders that is likely regulated by IL-1ra. This may be an important mechanism that protects the developing fetus from potentially deleterious consequences of maternal fever during pregnancy.     

Site-specific modulation of LPS-induced fever and interleukin-1 beta expression in rats by interleukin-10

Bacterial lipopolysaccharide (LPS) induces fever that is mediated by pyrogenic cytokines such as interleukin (IL)-1 beta. We hypothesized that the anti-inflammatory cytokine IL-10 modulates the febrile response to LPS by suppressing the production of pyrogenic cytokines. In rats, intravenous but not intracerebroventricular infusion of IL-10 was found to attenuate fever induced by peripheral administration of LPS (10 microg/kg iv). IL-10 also suppressed LPS-induced IL-1 beta production in peripheral tissues and in the brain stem. In contrast, central administration of IL-10 attenuated the febrile response to central LPS (60 ng/rat icv) and decreased IL-1 beta production in the hypothalamus and brain stem but not in peripheral tissues and plasma. Furthermore, intravenous LPS upregulated expression of IL-10 receptor (IL-10R1) mRNA in the liver, whereas intracerebroventricular LPS enhanced IL-10R1 mRNA in the hypothalamus. We conclude that IL-10 modulates the febrile response by acting in the periphery or in the brain dependent on the primary site of inflammation and that its mechanism of action most likely involves inhibition of local IL-1 beta production.     

Honokiol inhibits LPS-induced maturation and inflammatory response of human monocyte-derived dendritic cells

Honokiol (HNK) is a phenolic compound isolated from the bark of houpu (Magnolia officinalis), a plant widely used in traditional Chinese and Japanese medicine. While substantial evidence indicates that HNK possesses anti-inflammatory activity, its effect on dendritic cells (DCs) during the inflammatory reaction remains unclear. The present study investigates how HNK affects lipopolysaccharide (LPS)-stimulated human monocyte-derived DCs. Our experimental results show that HNK inhibits the inflammatory response of LPS-induced DCs by (1) suppressing the expression of CD11c, CD40, CD80, CD83, CD86, and MHC-II on LPS-activated DCs, (2) reducing the production of TNF-α, IL-1β, IL-6, and IL-12p70 but increasing the production of IL-10 and TGF-β1 by LPS-activated DCs, (3) inhibiting the LPS-induced DC-elicited allogeneic T-cell proliferation, and (4) shifting the LPS-induced DC-driven Th1 response toward a Th2 response. Further, our results show that HNK inhibits the phosphorylation levels of ERK1/2, p38, JNK1/2, IKKα, and IκBα in LPS-activated DCs. Collectively, the findings show that the anti-inflammatory actions of HNK on LPS-induced DCs are associated with the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways.     

Desipramine administered chronically inhibits lipopolysaccharide-stimulated production of IL-1β in the brain and plasma of rats

Nowadays, it is assumed that therapeutic efficacy of antidepressants depends, at least partly, on their anti-inflammatory properties. The present study investigated for the first time the effect of 21-day oral administration of desipramine on the lipopolysaccharide (LPS)-stimulated IL-1β concentration in the olfactory bulb, hypothalamus, frontal cortex, hippocampus and plasma of rats, and on the LPS-induced IL-1β mRNA level in the olfactory bulb. Desipramine (15 mg/kg/day) reduced significantly the LPS (250 μg/kg i.p.)-induced IL-1β concentration in the olfactory bulb, hypothalamus and in plasma, and diminished the LPS effect on IL-1β mRNA in the olfactory bulb. Plasma concentration of desipramine was comparable to its therapeutic range. By using the α1/α2-adrenoceptor antagonist prazosin and the unspecific β-adrenoceptor antagonist propranolol given prior to LPS, we found that the effect of desipramine on LPS-induced IL-1β production was partially mediated by both adrenoceptors in the olfactory bulb and plasma, and that β-adrenoceptors contributed also to its effect on the stimulated IL-1β concentration in the hypothalamus. The effect of LPS on the cerebral IL-1β levels was, in part, mediated by β-adrenoceptors and, in a region-specific manner, by α1/α2-adrenoceptors. The findings provide evidence for central and peripheral anti-inflammatory activity of desipramine and confirm the impact of the noradrenergic system on IL-1β production induced by an immunostimulatory challenge.     

Role of interleukin-4 in regulation of age-related inflammatory changes in the hippocampus

It is well documented that long term potentiation (LTP) is impaired in the hippocampus of the aged animal. Among the changes that contribute to this impairment is an increase in hippocampal concentration of the pro-inflammatory cytokine interleukin-1beta (IL-1beta), and increased IL-1beta-induced signaling. In this study we investigated the possibility that these changes were a consequence of decreased concentration of the anti-inflammatory cytokine, IL-4, and decreased IL-4-stimulated signaling. We report that functional IL-4 receptors are expressed on granule cells of the dentate gyrus and that receptor activation results in phosphorylation of JAK1 and STAT6. Hippocampal IL-4 concentration was decreased with age, and this was accompanied by a decrease in phosphorylation of JAK1 and STAT6. The evidence indicates that IL-4 modulates expression of IL-1beta mRNA and protein and that it attenuates IL-1beta-induced impairment of LTP and phosphorylation of JNK and c-Jun. We argued that, if a decrease in hippocampal IL-4 concentration significantly contributed to the age-related impairment in LTP, then restoration of IL-4 should restore LTP. To test this, we treated rats with VP015 (phospholipid microparticles-incorporating phosphatidylserine), which increases IL-4 concentration in hippocampus. The data indicate that the VP015-induced increase in IL-4 concentration in hippocampus of aged rats and lipopolysaccharide (LPS)-treated rats was accompanied by a reversal of the age-related and LPS-induced impairment in LTP in perforant path granule cell synapses. We propose that interplay between pro-inflammatory and anti-inflammatory responses impact significantly on synaptic function in the hippocampus of the aged rat.     

Moxifloxacin but not ciprofloxacin or azithromycin selectively inhibits IL-8, IL-6, ERK1/2, JNK, and NF-kappaB activation in a cystic fibrosis epithelial cell line

Cystic fibrosis (CF) is associated with severe neutrophilic airway inflammation. We showed that moxifloxacin (MXF) inhibits IL-8 and MAPK activation in monocytic and respiratory epithelial cells. Azithromycin (AZM) and ciprofloxacin (CIP) are used clinically in CF. Thus we now examined effects of MXF, CIP, and AZM directly on CF cells. IB3, a CF bronchial cell line, and corrected C38 cells were treated with TNF-alpha, IL-1beta, or LPS with or without 5-50 microg/ml MXF, CIP, or AZM. IL-6 and IL-8 secretion (ELISA), MAPKs ERK1/2, JNK, p38, and p65 NF-kappaB (Western blot) activation were measured. Baseline IL-6 was sixfold higher in IB3 than C38 cells but IL-8 was similar. TNF-alpha and IL-1beta increased IL-6 and IL-8 12- to 67-fold with higher levels in IB3 than C38 cells post-TNF-alpha (P < 0.05). Levels were unchanged following LPS. Baseline phosphorylated form of ERK1/2 (p-ERK1/2), JNK, and NF-kappaB p65 were higher in IB3 than C38 cells (5-, 1.4-, and 1.4-fold), and following TNF-alpha increased, as did the p-p38, by 1.6- to 2-fold. MXF (5-50 microg/ml) and CIP (50 microg/ml), but not AZM, suppressed IL-6 and IL-8 secretion by up to 69%. MXF inhibited TNF-alpha-stimulated MAPKs ERK1/2, 46-kDa JNK, and NF-kappaB up to 60%, 40%, and 40%, respectively. In contrast, MXF did not inhibit p38 activation, implying a highly selective pretranslational effect. In conclusion, TNF-alpha and IL-1beta induce an exaggerated inflammatory response in CF airway cells, inhibited by MXF more than by CIP or AZM. Clinical trials are recommended to assess efficacy in CF and other chronic lung diseases.     

Endogenous opioids: role in prostaglandin-dependent and -independent fever

This study evaluated the participation of mu-opioid-receptor activation in body temperature (T(b)) during normal and febrile conditions (including activation of heat conservation mechanisms) and in different pathways of LPS-induced fever. The intracerebroventricular treatment of male Wistar rats with the selective opioid mu-receptor-antagonist cyclic d-Phe-Cys-Try-d-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP; 0.1-1.0 microg) reduced fever induced by LPS (5.0 microg/kg) but did not change T(b) at ambient temperatures of either 20 degrees C or 28 degrees C. The subcutaneous, intracerebroventricular, and intrahypothalamic injection of morphine (1.0-10.0 mg/kg, 3.0-30.0 microg, and 1-100 ng, respectively) produced a dose-dependent increase in T(b). Intracerebroventricular morphine also produced a peripheral vasoconstriction. Both effects were abolished by CTAP. CTAP (1.0 microg icv) reduced the fever induced by intracerebroventricular administration of TNF-alpha (250 ng), IL-6 (300 ng), CRF (2.5 microg), endothelin-1 (1.0 pmol), and macrophage inflammatory protein (500 pg) and the first phase of the fever induced by PGF(2alpha) (500.0 ng) but not the fever induced by IL-1beta (3.12 ng) or PGE(2) (125.0 ng) or the second phase of the fever induced by PGF(2alpha). Morphine-induced fever was not modified by the cyclooxygenase (COX) inhibitor indomethacin (2.0 mg/kg). In addition, morphine injection did not induce the expression of COX-2 in the hypothalamus, and CTAP did not modify PGE(2) levels in cerebrospinal fluid or COX-2 expression in the hypothalamus after LPS injection. In conclusion, our results suggest that LPS and endogenous pyrogens (except IL-1beta and prostaglandins) recruit the opioid system to cause a mu-receptor-mediated fever.     

Modulation of TLR4 signaling by a novel adaptor protein signal-transducing adaptor protein-2 in macrophages

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin and Src homology 2-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies have demonstrated that STAP-2 binds to STAT3 and STAT5, and regulates their signaling pathways. In the present study, STAP-2 was found to positively regulate LPS/TLR4-mediated signals in macrophages. Disruption of STAP-2 resulted in impaired LPS/TLR4-induced cytokine production and NF-kappaB activation. Conversely, overexpression of STAP-2 enhanced these LPS/TLR4-induced biological activities. STAP-2, particularly its Src homology 2-like domain, bound to both MyD88 and IkappaB kinase (IKK)-alphabeta, but not TNFR-associated factor 6 or IL-1R-associated kinase 1, and formed a functional complex composed of MyD88-STAP-2-IKK-alphabeta. These interactions augmented MyD88- and/or IKK-alphabeta-dependent signals, leading to enhancement of the NF-kappaB activity. These results demonstrate that STAP-2 may constitute an alternative LPS/TLR4 pathway for NF-kappaB activation instead of the TNFR-associated factor 6-IL-1R-associated kinase 1 pathway.     

Cloning and characterization of two novel zebrafish P2X receptor subunits

Activation of Kupffer cells by lipopolysaccharide (LPS) after ethanol feeding results in overproduction of TNF-alpha, leading to liver injury. Since dilinoleoylphosphatidylcholine (DLPC) protects against liver injury and has antioxidant properties, we investigated whether it alters LPS signaling leading to decreased TNF-alpha production. Kupffer cells were isolated from rats fed alcohol-containing or isocaloric control diets for 3 weeks. With ethanol, cytochrome P4502E1 was upregulated. When stimulated with LPS in culture, Kupffer cells released more TNF-alpha compared to control rats; DLPC diminished the increase. It also reduced ERK1/2 and p38 phosphorylation as well as NF-kappaB activation with decreased nuclear p65 and increased cytosolic IkappaB-alpha expression. ERK1/2 and NF-kappaB activation were abolished by the ERK1/2 inhibitor PD098059. The p38 inhibitor SB203580 abolished p38 activation without affecting NF-kappaB. Both inhibitors reduced TNF-alpha generation. Thus, DLPC diminishes LPS-dependent TNF-alpha generation by inhibiting p38 and ERK1/2 activation; the latter leads to decreased NF-kappaB activation.     

Implications of proteasome inhibition: an enhanced macrophage phenotype

The objective of this study was to elucidate the role of the cellular proteasome on endotoxin-mediated activation of the macrophage. To study this role, THP-1 cells were stimulated with lipopolysaccharide (LPS) with selective cells being pretreated with the proteasome inhibitor, lactacystin or MG-132. LPS stimulation led to the phosphorylation and degradation of IRAK, followed by activation of JNK/SAPK, ERK 1/2, and p38. Subsequently, LPS induced the degradation of IkappaB, and the nuclear activation of NF-kappaB and AP-1. Activation of these pathways was associated with the production of IL-6, IL-8, IL-10, and TNF-alpha. Proteasome inhibition with either lactacystin or MG-132 attenuated LPS-induced IRAK degradation, and enhanced activation of JNK/SAPK, ERK 1/2, and p38. Proteasome inhibition, also, led to increased LPS-induced AP-1 activation, and attenuated LPS-induced IkappaB degradation resulting in abolished NF-kappaB activation. Proteasome inhibition led to significant modulation of LPS-induced cytokine production; increased IL-10, no change in IL-6, and decreased IL-8, and TNF-alpha. Thus, this study demonstrates that cellular proteasome is critical to regulation of LPS-induced signaling within the macrophage, and inhibition of the proteasome results in a conversion to an anti-inflammatory phenotype.