Immuno-metabolic impact of the multiple sclerosis patients' sera on endothelial cells of the blood-brain barrier.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease which results from the invasion of the brain by activated immune cells across the endothelial cells (ECs) of the blood-brain barrier (BBB), due to loss of immune self-tolerance. Many reports define the metabolic profile of immune cells in MS, however little is known about the metabolism of the BBB ECs during the disease. We aim to determine whether circulating factors in MS induce metabolic alterations of the BBB ECs compared to a healthy state, which can be linked with disruption of BBB integrity and subsequent immune cell extravasation. METHODS AND RESULTS: In this report, we used an in vitro model to study the effect of sera from naïve-to-treatment, relapsing-remitting MS (RRMS) patients on the human brain microvascular endothelium, comparing effects to age/sex-matched healthy donor (HD) sera. Our data show that RRMS serum components affect brain endothelial cells by impairing intercellular tightness through the down-modulation of occludin and VE-cadherin, and facilitating immune cell extravasation through upregulation of intercellular adhesion molecules (ICAM-1) and P-glycoprotein (P-gp). At a metabolic level, the treatment of the endothelial cells with RRMS sera reduced their glycolytic activity (measured through the extracellular acidification rate-ECAR) and oxygen consumption rate (oxidative phosphorylation rate-OCR). Such changes were associated with the down-modulation of endothelial glucose transporter 1 (GLUT-1) expression and by altered mitochondrial membrane potential. Higher level of reactive oxygen species released from the endothelial cells treated with RRMS sera indicate a pro-inflammatory status of the cells together with the higher expression of ICAM-1, endothelial cell cytoskeleton perturbation (stress fibres) as well as disruption of the cytoskeleton signal transduction MSK1/2 and ß-catenin phosphorylation. CONCLUSIONS: Our data suggest that circulating factors present in RRMS patient serum induce physiological and biochemical alterations to the BBB, namely reducing expression of essential tightness regulators, as well as reduced engagement of glycolysis and alteration of mitochondrial potential. As these last changes have been linked with alterations in nutrient usage and metabolic function in immune cells; we propose that the BBB endothelium of MS patients may similarly undergo metabolic dysregulation, leading to enhanced permeability and increased disease susceptibility.

Estrogen protects the blood-brain barrier from inflammation-induced disruption and increased lymphocyte trafficking.

Sex differences have been widely reported in neuroinflammatory disorders, focusing on the contributory role of estrogen. The microvascular endothelium of the brain is a critical component of the blood-brain barrier (BBB) and it is recognized as a major interface for communication between the periphery and the brain. As such, the cerebral capillary endothelium represents an important target for the peripheral estrogen neuroprotective functions, leading us to hypothesize that estrogen can limit BBB breakdown following the onset of peripheral inflammation. Comparison of male and female murine responses to peripheral LPS challenge revealed a short-term inflammation-induced deficit in BBB integrity in males that was not apparent in young females, but was notable in older, reproductively senescent females. Importantly, ovariectomy and hence estrogen loss recapitulated an aged phenotype in young females, which was reversible upon estradiol replacement. Using a well-established model of human cerebrovascular endothelial cells we investigated the effects of estradiol upon key barrier features, namely paracellular permeability, transendothelial electrical resistance, tight junction integrity and lymphocyte transmigration under basal and inflammatory conditions, modeled by treatment with TNFα and IFNγ. In all cases estradiol prevented inflammation-induced defects in barrier function, action mediated in large part through up-regulation of the central coordinator of tight junction integrity, annexin A1. The key role of this protein was then further confirmed in studies of human or murine annexin A1 genetic ablation models. Together, our data provide novel mechanisms for the protective effects of estrogen, and enhance our understanding of the beneficial role it plays in neurovascular/neuroimmune disease.

Formyl peptide receptors and the regulation of ACTH secretion: targets for annexin A1, lipoxins, and bacterial peptides.

The N-formyl peptide receptors (FPRs) are a family of G-protein coupled receptors that respond to proinflammatory N-formylated bacterial peptides (e.g., formyl-Met-Leu-Phe, fMLF) and, thus, contribute to the host response to bacterial infection. Paradoxically, a growing body of evidence suggests that some members of this receptor family may also be targets for certain anti-inflammatory molecules, including annexin A1 (ANXA1), which is an important mediator of glucocorticoid (GC) action. To explore further the potential role of FPRs in mediating ANXA1 actions, we have focused on the pituitary gland, where ANXA1 has a well-defined role as a cell-cell mediator of the inhibitory effects of GCs on the secretion of corticotrophin (ACTH), and used molecular, genetic, and pharmacological approaches to address the question in well-established rodent models. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis identified mRNAs for four FPR family members in the mouse anterior pituitary gland, Fpr-rs1, Fpr-rs2, Fpr-rs6, and Fpr-rs7. Functional studies confirmed that, like dexamethasone, ANXA1 and two ANXA1-derived peptides (ANXA1(1-188) and ANXA1(Ac2-26)) inhibit the evoked release of ACTH from rodent anterior pituitary tissue in vitro. Fpr1 gene deletion failed to modify the pituitary responses to dexamethasone or ANXA1(Ac2-26). However, lipoxin A4 (LXA4, 0.02-2 microM, a lipid mediator with high affinity for Fpr-rs1) mimicked the inhibitory effects of ANXA1 on ACTH release as also did fMLF in high (1-100 microM) but not lower (10-100 nM) concentrations. Additionally, a nonselective FPR antagonist (Boc1, 100 microM) overcame the effects of dexamethasone, ANXA1(1-188), ANXA1(Ac2-26), fMLF, and LXA4 on ACTH release, although at a lower concentration (50 microM), it was without effect. Together, the results suggest that the actions of ANXA1 in the pituitary gland are independent of Fpr1 but may involve other FPR family members, in particular, Fpr-rs1 or a closely related receptor. They thus provide the first evidence for a role of the FPR family in the regulation of neuroendocrine function.

Post-translational modification plays an essential role in the translocation of annexin A1 from the cytoplasm to the cell surface.

Annexin A1 (ANXA1) has an important role in cell-cell communication in the host defense and neuroendocrine systems. In both systems, its actions are exerted extracellularly via membrane-bound receptors on adjacent sites after translocation of the protein from the cytoplasm to the cell surface of adjacent cells. This study used molecular, microscopic, and pharmacological approaches to explore the mechanisms underlying the cellular exportation of ANXA1 in TtT/GF (pituitary folliculo-stellate) cells. LPS caused serine-phosphorylation of ANXA1 (ANXA1-S27-PO4) and translocation of the phosphorylated protein to the cell membrane. The fundamental requirement of phosphorylation for membrane translocation was confirmed by immunofluorescence microscopy on cells transfected with wild-type or mutated (S27/A) ANXA1 constructs tagged with enhanced green fluorescence protein. The trafficking of ANXA1-S27-PO4 to the cell surface was dependent on PI3-kinase and MAP-kinase. It also required HMG-coenzyme A and myristoylation. The effects of HMG-coenzyme A blockade were overcome by mevalonic acid (the product of HMG-coenzyme A) and farnesyl-pyrophosphate but not by geranyl-geranylpyrophosphate or cholesterol. Together, these results suggest that serine-27 phosphorylation is essential for the translocation of ANXA1 across the cell membrane and also identify a role for isoprenyl lipids. Such lipids could target consensus sequences in ANXA1. Alternatively, they may target other proteins in the signal transduction cascade (e.g., transporters).

Annexin-1 downregulation in thyroid cancer correlates to the degree of tumor differentiation.

We investigated the expression of annexin-1 (ANXA1) in thyroid carcinoma cell lines and in thyroid cancers with a different degree of differentiation. The highest level of ANXA1 expression examined by Western blotting was detected in the papillary carcinoma cells (NPA) and in the follicular cells (WRO). On the other hand, the most undifferentiated thyroid carcinoma cells (ARO and FRO) presented the lowest level of ANXA1 expression. In surgical tissue specimens from 32 patients with thyroid cancers, we found high immunoreactivity for ANXA1 in papillary (PTC) and follicular (FTC) thyroid cancers while in undifferentiated thyroid cancers (UTC) the expression of the protein was barely detectable. Control thyroid tissue resulted positive for ANXA1. In summary, 70% of UTC examined weakly expressed ANXA1, whereas 65% of PTC or FTC specimens tested showed high expression of the protein. Thus ANXA1 expression may correlate with the tumorigenesis suggesting that the protein may represent an effective differentiation marker in thyroid cancer.

Dexamethasone induces the expression of the mRNA of lipocortin 1 and 2 and the release of lipocortin 1 and 5 in differentiated, but not undifferentiated U-937 cells.

The effect of dexamethasone on mRNA and protein synthesis of lipocortins (LCT) 1, 2 and 5 has been investigated in U-937 cells. A constitutive expression of both mRNAs and proteins was detected in undifferentiated U-937 cells. This constitutive level was increased time- and dose-dependently by incubation with phorbol myristate acetate (PMA). In U-937 cells differentiated by 24 h incubation with 6 ng/ml PMA, dexamethasone (DEX) (1 microM for 16 h) caused an increased synthesis of the mRNA level of LCT-1 and 2, but not of LCT-5, over the level induced by PMA. DEX had no effect in undifferentiated cells. Moreover, DEX stimulated the extracellular release of LCT-1 and 5, but not of LCT-2, and inhibited the release of PGE2 and TXB2 only in the differentiated U-937 cells. These results suggest that the responsiveness of these cells to glucocorticoids is dependent on the phase of cell differentiation. The selective release of lipocortins by differentiated U-937 cells may explain, at least in part, the inhibition by DEX of the prostanoid release.

Modulation of phospholipase A2 activity in human fibroblasts.

1. Human embryonic skin fibroblasts (HSF) incubated overnight with either human recombinant interleukin-1 alpha (rIL-1 alpha) or interleukin-1 beta (rIL-1 beta) released large amounts of prostaglandin E2 (PGE2). 2. rIL-1 beta, bradykinin (Bk) and arachidonic acid (AA) significantly stimulated PGE2 release from HSF incubated overnight in the presence of either interleukin. 3. Hydrocortisone inhibited the PGE2 release induced by rIL-1 beta and Bk, but not by AA. 4. The steroid inhibitory effect was reversed by actinomycin D as well as by an anti-lipocortin monoclonal antibody. 5. The results suggest that in HSF, rIL-1 beta is able to stimulate both cyclo-oxygenase and phospholipase A2 (PLA2) activity. 6. The stimulation of PLA2 activity by rIL-1 beta is inhibited by hydrocortisone, probably via induction of lipocortin-like proteins.

Dexamethasone-induced translocation of lipocortin (annexin) 1 to the cell membrane of U-937 cells.

Lipocortin (annexin) 1 is a putative mediator of the inflammatory effects of glucocorticoids. By flow cytometric analysis (FACS) we have studied the effect of dexamethasone on the cellular localization of lipocortin 1. U-937 cells were incubated with or without 10 nM phorbol 12-myristate 13-acetate (PMA) to induce cell differentiation. Then 1 microM dexamethasone was added and incubation carried out for increasing times (1-24 h). Dexamethasone caused a time-dependent biphasic translocation of lipocortin 1 from the intracellular compartment to the cell membrane with maximal membrane expression at 4 and 24 h. In differentiated U-937 cells the steroid-induced membrane accumulation of lipocortin 1 was significantly higher than that of undifferentiated cells. The accumulation of the protein in the cell membrane may precede its release which is stimulated by dexamethasone in differentiated U-937 cells. Since extracellular lipocortin 1 has anti-inflammatory properties the modulation of the translocation/secretion process of the protein by glucocorticoids may be part of their mechanism of action.

A novel anti-inflammatory peptide from human lipocortin 5.

1. A novel anti-inflammatory peptide (residues 204-212) of human recombinant lipocortin 5 (hrLC5) found on the high similarity region with uteroglobin is described. 2. Peptide 204-212 dose-dependently inhibited the contractions of rat isolated stomach strips elicited by porcine pancreatic phospholipase A2 (PLA2). Contractions caused by arachidonic acid (AA), prostaglandin E2 (PGE2) and 5-hydroxytryptamine were not affected. No direct enzyme inhibition was observed in a radiochemical assay. 3. PGE2 release by both human fibroblasts and rat macrophages was reduced by peptide 204-212 in a dose-dependent manner. 4. The development of carrageenin-induced oedema in rats was significantly inhibited by the local administration of peptide 204-212. 5. The pattern and potency of the biological effects of peptide 204-212 are similar to those of antiflammin 2, a lipocortin 1-derived peptide. 6. It is suggested that peptide 204-212 may represent the active site responsible for the anti-inflammatory properties of lipocortin 5.

U937 cells deprived of endogenous annexin 1 demonstrate an increased PLA2 activity.

Annexin 1 (An 1), a phospholipid and calcium binding protein, is strongly expressed in differentiated U 937 cells. In attempting to correlate the expression of An 1 with phospholipase A2 (PLA2) activity, U 937 cells were stably transfected both with a Sense and Antisense cDNA for An 1. PLA2 activity was measured by Flow cytometry analysis utilizing the bis-Bodipy-C11-PC fluorescent probe. U 937 cells stably transfected with the sense or antisense vectors were differentiated for 24 h with phorbol 12-myristate 13-acetate (PMA, 6 ng ml(-1)). Both in undifferentiated and differentiated cells, the Antisense clone (36.4 AS) showed consistently higher PLA2 activity than the control Sense clone (15 S). Since the fluorescent probe measures the total PLA2 activity, we used two different stimuli, PMA: (100 ng ml(-1)) or lipopolysaccharide (LPS, 10 ng ml(-1)), and two different inhibitors, to discriminate the PLA2 involved (namely arachidonyl trifluoromethyl ketone or AACOCF3, which is specific for the cytosolic PLA2, and SB 203347 specific for the secretory PLA2). In the Antisense clone the inhibitory effect of AACOCF was stronger [68%, P<0.025] than in the Sense, which may reflect the lower endogenous level of An 1 present in the cells. On the contrary, the inhibitory effect of SB 203347 [60% of inhibition] was identical in both clones. Since cPLA2 activity is correlated with its phosphorylation, Western and shift blot analysis were performed. They did not show any significative difference between the phosphorylated and non phosphorylated form of the enzyme in both the differentiated or not, Sense and Antisense clones. Furthermore the tyrosine phosphorylation analysis of An 1 showed that less than 10% of An 1 was phosphorylated irrespective of PMA presence or absence. From the pattern of inhibition observed, we propose that the endogenous unphosphorylated form of An 1 may act intracellularly to block the activity of a cytosolic PLA2.

Transfection of annexin 1 in monocytic cells produces a high degree of spontaneous and stimulated apoptosis associated with caspase-3 activation.

Transfection of the pre-monomyelocytic U937 cell line with a plasmid coding for full-length annexin 1 (ANX1, 347 amino acid) leads to cell death by promoting apoptosis. In addition, over-expression of the N-terminal and the first domain of the protein (144 amino acids, clone ANX1-S), which does not contain the Ca2+ binding sites, gives susceptibility to cell apoptosis following activation by either 5 ng ml(-1) tumour necrosis factor (TNF)-alpha or 1 - 40 microg ml-1 etoposide. This was demonstrated by using the fluorescent labelled annexin V, cell cycle and nuclear staining analyses. Transfection with an empty plasmid (clone CMV) or with a plasmid carrying the cDNA antisense for ANX1 (clone ANX1-AS) did not alter U937 cells to the degree of apoptosis promoted by either stimulant. Treatment of CMV U937 cells with TNF-alpha increased ANX1 mRNA and protein expression in a time-dependent manner, with maximal increases at 3 and 6 h, respectively. Clone ANX1-S showed higher constitutive (more than 2 fold) and activated caspase-3 activity, associated with higher phospholipase A2 (PLA2) activity (in the region of +50 - 100%), whereas expression of cytosolic PLA2 Bax and Bcl-2 were similar in all cell clones, as determined by Western blotting. In conclusion, this study demonstrates a complex regulatory role of cell apoptosis for ANX1, at least with regards to cells of the myelo-monocytic lineage.

Anti-inflammatory mechanism of alminoprofen: action on the phospholipid metabolism pathway.

Alminoprofen is a nonsteroidal anti-inflammatory drug (NSAID) of the phenylpropionic acid class. It has anti-inflammatory properties different from the classical NSAID. Using both in vitro systems of cells in culture and in vivo models of inflammation, we report here that alminoprofen possesses both antiphospholipase A2 (PLA2) activity and anti-cycloxygenase (COX) activity. The PLA2 targeted by alminoprofen is likely the secretory phospholipase A2 (sPLA2) while the COX targeted is the COX-2.

Evidence from studies on co-cultures of TtT/GF and AtT20 cells that Annexin 1 acts as a paracrine or juxtacrine mediator of the early inhibitory effects of glucocorticoids on ACTH release.

Annexin 1 (ANXA1) is a key mediator of the inhibitory effects of glucocorticoids on adrenocorticotropic hormone (ACTH) release, which develop within 1-2 h of a steroid challenge. Our previous studies, which showed that (i) ANXA1 is expressed principally by the nonsecretory folliculo-stellate cells in the pituitary gland; (ii) glucocorticoids cause the exportation of ANXA1 from these cells; and (iii) corticotrophs express specific ANXA1 binding sites, led us to propose that ANXA1 serves as a paracrine or juxtacrine mediator of glucocorticoids. To address this hypothesis, we examined ANXA1-dependent glucocorticoid actions in co-cultures of murine corticotroph (AtT20 clone D1) and folliculo-stellate (TtT/GF) cell lines. ANXA1 mRNA and protein were found in abundance in TtT/GF cells but neither was detectable in the AtT20 cells. AtT20 cells (alone and in co-culture with TtT/GF cells) responded to corticotropin-releasing hormone (CRH) (0.1-1 micro m) with increased ACTH release. The CRH-stimulated release of ACTH from AtT20 cells cultured alone was unaffected by preincubation with dexamethasone (Dex, 100 nm); by contrast, in co-cultures of AtT20 and TtT/GF cells, the steroid readily inhibited the secretory response to CRH. The effects of Dex on ACTH release were mimicked by N-terminal ANXA1 fragments (ANXA1Ac2-26, 2 micro g/ml and ANXA11-188, 0.1 ng/ml) and reversed by mifepristone (1 micro m) and by an antisense oligodeoxynucleotide (ODN) to ANXA1 (50 nm) but not by control ODNs. The antisense ODN also specifically blocked the Dex-induced externalization of ANXA1 from TtT/GF cells. Immunofluorescence imaging of the co-cultures localized the exported protein to the vicinity of the AtT20 cells and identified ANXA1 binding sites on these cells. These results provide functional and histological evidence to support our premise that the early inhibitory effects of glucocorticoids on ACTH release are dependent upon paracrine/juxtacrine actions of ANXA1 derived from folliculo-stellate cells.

Kinase-dependent regulation of the secretion of thyrotrophin and luteinizing hormone by glucocorticoids and annexin 1 peptides.

Our previous studies have identified a role for annexin 1 (ANXA1), a protein produced by the pituitary folliculostellate cells, as a paracrine/juxtacrine mediator of the acute regulatory effects of glucocorticoids on the release of adrenocorticotropic hormone and other pituitary hormones. In the present study, we focused on the secretion of thyroid stimulating hormone (TSH) and luteinizing hormone (LH) and used a battery of ANXA1-derived peptides to identify the key domains in the ANXA1 molecule that are critical to the inhibition of peptide release. In addition, as ANXA1 is a substrate for protein kinase C (PKC) and tyrosine kinase, we examined the roles of these kinases in the manifestation of the ANXA1-dependent inhibitory actions of dexamethasone on TSH and LH release. Dexamethasone suppressed the forskolin-induced release of TSH and LH from rat anterior pituitary tissue in vitro. Its effects were mimicked by human recombinant ANXA1 (hrANXA1) and a truncated protein, ANXA1(1-188). ANXA1(Ac2-26), also suppressed stimulated peptide release but it lacked both the potency and the efficacy of the parent protein. Shorter N-terminal ANXA1 sequences were without effect. The PKC inhibitor PKC(19-36) abolished the inhibitory actions of dexamethasone on the forskolin-evoked release of TSH and LH; it also attenuated the inhibitory actions of ANXA1(Ac2-26). Similar effects were produced by annexin 5 (ANXA5) which sequesters PKC in other systems. By contrast, the tyrosine kinase inhibitors, p60v-src (137-157) and genistein, had no effect on the secretion of TSH or LH alone or in the presence of forskolin and/or dexamethasone. Dexamethasone caused the translocation of a tyrosine-phosphorylated species of ANXA1 to the surface of pituitary cells. The total amount of ANXA1 exported from the cells in response to the steroid was unaffected by tyrosine kinase blockade. However, the degree of tyrosine-phosphorylation of the exported protein was markedly reduced by genistein. These results suggest that (i) the ANXA1-dependent inhibitory actions of dexamethasone on the release of TSH and LH require PKC and sequences in the N-terminal domain of ANXA1, but are independent of tyrosine kinase, and (ii) while dexamethasone induces the cellular exportation of a tyrosine-phosphorylated species of ANXA1, tyrosine phosphorylation per se is not critical to the steroid-induced passage of ANXA1 across the membrane.

Macrophage-specific eicosanoid synthesis inhibition and lipocortin-1 induction by glucocorticoids.

It has been suggested that the induction of lipocortin-1, a phospholipase A2-inhibitory protein, may mediate the anti-inflammatory action of glucocorticoids. We assessed the production of prostaglandin E2, thromboxane B2, and leukotriene B4 and the expression of lipocortin-1 in different populations of blood leukocytes and in alveolar macrophages (obtained by bronchoalveolar lavage) from patients with inflammatory lung diseases (bronchial asthma, n = 21; interstitial lung disease, n = 6) undergoing glucocorticoid treatment at clinically effective doses. No inhibition of eicosanoid production was observed in either whole blood or single populations of blood leukocytes (granulocytes and monocytes) stimulated with ionophore A-23187, N-formyl-L-methionyl-L-leucyl-L-phenylalanine, or zymosan. Conversely, eicosanoid production from alveolar macrophages (assessed in 9 cases) was significantly inhibited by glucocorticoids. After ionophore stimulation, eicosanoid production was as follows (in ng/ml): prostaglandin E2, 0.19 +/- 0.06 and 0.06 +/- 0.01; thromboxane B2, 2.9 +/- 0.9 and 0.5 +/- 0.1; leukotriene B4, 6.6 +/- 1.1 and 3.6 +/- 1.0, before and after treatment, respectively (P < 0.05 for all differences). Lipocortin-1 expression, determined by Western blot and enzyme immunoassay, was significantly (P < 0.05) stimulated in alveolar macrophages, but not in blood leukocytes, by glucocorticoid treatment. These results indicate that alveolar macrophages, at variance from blood leukocytes, are the most likely cell target for glucocorticoid-induced eicosanoid inhibition and lipocortin expression. We suggest that cell responsiveness to glucocorticoids is acquired during differentiation from monocyte to tissue macrophage.

Mechanism of acute toxicity of IL-1 beta in mice.

Human recombinant IL-1 beta was able to kill C3H/HeJ mice only when inoculated intravenously at very high doses. IL-1 beta, inoculated at 100 mg/kg i.v. as a bolus, induced a shock-like state characterized by anorexia, severe hypothermia and hypoglycemia and persistent neutrophilia, leading to death in 55% of animals generally between 24 and 48 h. In contrast, the noninflammatory adjuvant IL-1 beta peptide VQGEESNDK (position 163-171) did not induce any toxic effect in vivo, when administered following the same schedule. At variance with what was previously observed in endotoxin induced shock, IL-1 beta induced death was not preceded by appearance of circulating TNF. On the other hand, very high and persistent levels of circulating IL-6 could be detected after lethal IL-1 beta administration. Treatment of mice with ibuprofen or with chlorpromazine, both known to counteract some of the toxic effects of IL-1 in vivo, could protect from IL-1 beta induced mortality. Both drugs, at doses protecting from IL-1 beta induced death, were able to abolish IL-1 beta-induced rise of circulating phospholipase A2 (PLA2) activity, and the subsequent generation of toxic PLA2-derived metabolites.

Increased apoptosis in U937 cells over-expressing lipocortin 1 (annexin I).

The potential involvement of endogenous lipocortin 1 in the process of cellular apoptosis, particularly in cells of the myelo-monocytic lineage, has been investigated. U937 cells were transfected either with an antisense or a sense DNA for lipocortin 1 and the stable clones 36.4AS clone (20-40% lower lipocortin 1 levels) and 15S (30% higher lipocortin 1 levels) were obtained. Cell apoptosis was induced by incubation with tumor necrosis factor-alpha: optimal responses were observed within a 24 h incubation period at a 5 ng/ml concentration. Apoptosis was assessed both morphologically, by annexin V binding and cell cycle analysis with propidium iodide. Whilst no consistent difference was seen between wild type cells and clone 36.4AS, a higher incidence of apoptosis (ranging from +30% to + 60%) was observed in the 15S clone. Release of arachidonic acid from loaded cells was promoted by 24 h incubation with the cytokine, and a higher degree of release was measured in the 15S clone. These data indicate that endogenous intracellular lipocortin 1 is involved in the promotion of apoptosis in cells of the myelo-monocytic derivation.

Expression of annexin and annexin-mRNA in rat brain under influence of steroid drugs.

Brain tissue of rats pretreated with methylprednisolone or with the 21-aminosteroid U74389F, and that of untreated control rats, was assessed for the expression of Annexin-1 (Anx-1) and the transcription of its mRNA. For this purpose Anx-1 cDNA was amplified and simultaneously a T7-RNA-polymerase promotor was incorporated into the cDNA using Polymerase Chain Reaction (PCR). Then digoxigenin-11-UTP was incorporated into the transcribed cRNA with T7-RNA-polymerase. With this probe in situ hybridization was carried out in sections of the brain. The probe was visualized by an immunoassay using an anti-digoxigenin antibody conjugate. Anx-1 protein was assessed by means of immunohistochemistry using a polyclonal antibody. The various brain areas of the control animals showed an appreciable amount of Anx-1 at mRNA or protein level; on the other hand, the animals which had been pretreated with either steroid, showed a more intense Anx-1 mRNA signal than the controls in many areas. In the pretreated animals Anx-1 immunostaining was unchanged in cortex, basal ganglia, amygdala and septum, but more intense in hippocampus, hypothalamus and thalamus. In ependyma, choroid plexus, meninges, and vascular walls there was no Anx-1 mRNA transcription detectable. An opposite profile was shown by the Anx-1 immunoreactivity, the protein was present in control animals as well as the steroid-pretreated animals, suggesting that here the protein was either from systemic origin, or has diffused from adjacent structures. The results indicate that Anx-1 mRNA transcription is upregulated by either steroid, and that in the untreated animals there is a resting level of Anx-1 mRNA transcription, presumably reflecting physiological influences on Anx-1 expression.

Studies on the induction of lipocortin-1 by glucocorticoids.

Part of the anti-inflammatory efficacy of glucocorticoids has been ascribed to the inhibition of eicosanoid formation which is brought about by lipocortins (LC, phospholipase A2 inhibitory proteins) whose synthesis is induced by corticosteroids. We have investigated the effect of glucocorticoids on eicosanoid formation and lipocortin-1 induction in a human cell line in vitro and in patients with inflammatory lung disease in vivo. Human promyelocitic U-937 cell line was differentiated by 24 h incubation with phorbol myristate acetate (PMA, 6 ng/ml), then dexamethasone (1 microM) was added for further 16 h. In these conditions the steroid treatment caused both the release of lipocortin-1 in the cellular supernatant and the inhibition of eicosanoid release. These results suggest that the responsiveness of these cells to steroids is dependent on the phase of cell activation-differentiation. The selective release of the lipocortin-1 may explain the inhibition of eicosanoid formation. Patients with inflammatory lung disease underwent glucocorticoid treatment at clinically effective doses. LC-1 expression was significantly stimulated in alveolar macrophages, but not in blood-derived lympho-monocytes. These results suggest that also in vivo cell responsiveness to glucocorticoids is acquired during cell differentiation from blood monocyte to tissue macrophage.

Evidence from in vitro and in vivo studies showing that nuclear factor-κB within the pituitary folliculostellate cells and corticotrophs regulates adrenocorticotrophic hormone secretion in experimental endotoxaemia.

The hypothalamic-pituitary-adrenocortical (HPA) responses to bacterial infection are mediated, in part, by the actions of lipopolysaccharide (LPS) on pituitary folliculostellate (FS) cells that release pro-inflammatory cytokines [e.g. interleukin (IL)-6] and thereby facilitate adrenocorticotrophic hormone (ACTH) release from neighbouring corticotrophs. In the present study, two murine pituitary cell lines [TtT/GF (FS cells) and AtT20 D16:16 (corticotrophs)], alone and in co-culture, and an in vivo model of endotoxaemia were used to examine the potential role of nuclear factor-kappa B (NF-κB) in mediating LPS-induced ACTH secretion. Both cell lines expressed mRNAs for the key components of the LPS signalling system. LPS stimulated IL-6 release from TtT/GF cells via a glucocorticoid-sensitive, NF-κB-dependent mechanism; it also activated NF-κB in AtT20 cells, as did corticotrophin-releasing hormone (CRH). IL-6 potentiated (but LPS reduced) the stimulatory effects of CRH on ACTH release from AtT20 cells, whereas blockade of NF-κB (SC-514) increased the ACTH release induced by CRH in the presence or absence of LPS. In co-cultures, CRH and LPS acted synergistically to induce release of both IL-6 and ACTH. However, although SC-514 suppressed the release of IL-6 evoked by CRH and LPS, it potentiated the concomitant increase in ACTH release. In vivo both immunological (LPS) and psychological (restraint) stress increased intrapituitary NF-κB, whereas an NF-κB inhibitor (PHA781535E) attenuated the LPS-induced release of ACTH and abolished the HPA response to restraint stress. The results obtained in the present study support the premise that NF-κB plays an important role in mediating LPS signalling in the anterior pituitary gland, particularly in relation to IL-6 and ACTH secretion, and provide novel evidence that NF-κB blockade in vivo compromises stress-induced ACTH release.