Expression and Induction of Secretory Phospholipase A Group IB in Brain

Summary Secretory phospholipases A₂ (sPLA₂) form a diverse family of enzymes involved in physiologicand pathologic processes. Common among all sPLA₂ is the ability to cleave acyl groups of phospholipids at 2_C of the glycerol backbone, thereby releasingfatty acid and a lysophospholipid. Several sPLA₂ have been cloned and characterized in various tissues.Furthermore, receptors have been identified. In the nervous system sPLA₂ groups IIA, IIE, IIF, V, and XII have been identified, and binding sites for sPLA₂ group IB (sPLA₂-IB) have been found. Here, we report sPLA₂-IB in rat and human brain as well as in neurons in primary culture. The distribution of sPLA₂-IB seems to be mainly neuronal, with the highest abundance occurring in the cerebral cortex and hippocampus. We also find that genes encoding sPLA₂-IB are induced by kainic acid and by electroshock-induced convulsions.Based on the present results we suggest that sPLA₂-IB may be a neuronal intercellular signalling modulator.     

Cooperative role of endogenous leucotrienes and platelet‐activating factor in ischaemia–reperfusion‐mediated tissue injury

Insufficient oxygen delivery to organs leads to tissue dysfunction and cell death. Reperfusion, although vital to organ survival, initiates an inflammatory response that may both aggravate local tissue injury and elicit remote organ damage. Polymorphonuclear neutrophil (PMN) trafficking to remote organs following ischaemia/reperfusion (I/R) is associated with the release of lipid mediators, including leucotriene (LT) B₄, cysteinyl‐LTs (CysLTs) and platelet‐activating factor (PAF). Yet, their potentially cooperative role in regulating I/R‐mediated inflammation has not been thoroughly assessed. The present study aimed to determine the cooperative role of lipid mediators in regulating PMN migration, tissue oedema and injury using selective receptor antagonists in selected models of I/R and dermal inflammation. Our results show that rabbits, pre‐treated orally with BIIL 284 and/or WEB 2086 and MK‐0571, were protected from remote tissue injury following I/R or dermal inflammation in an additive or synergistic manner when the animals were pre‐treated with two drugs concomitantly. The functional selectivity of the antagonists towards their respective agonists was assessed in vitro, showing that neither BIIL 284 nor WEB 2086 prevented the inflammatory response to IL‐8, C5a and zymosan‐activated plasma stimulation. However, these agonists elicited LTB₄ biosynthesis in isolated rabbit PMNs. Similarly, a cardioprotective effect of PAF and LTB₄ receptor antagonists was shown following myocardial I/R in mice. Taken together, these results underscore the intricate involvement of LTB₄ and PAF in each other's responses and provide further evidence that targeting both LTs and PAF receptors provides a much stronger anti‐inflammatory effect, regulating PMN migration and oedema formation.     

The Role of Secretory Phospholipase A2 in the Central Nervous System and Neurological Diseases

Secretory phospholipase A₂ (sPLA₂s) are small secreted proteins (14–18 kDa) and require submillimolar levels of Ca²⁺ for liberating arachidonic acid from cell membrane lipids. In addition to the enzymatic function, sPLA₂ can exert various biological responses by binding to specific receptors. Physiologically, sPLA₂s play important roles on the neurotransmission in the central nervous system and the neuritogenesis in the peripheral nervous system. Pathologically, sPLA₂s are involved in the neurodegenerative diseases (e.g., Alzheimer’s disease) and cerebrovascular diseases (e.g., stoke). The common pathology (e.g., neuronal apoptosis) of Alzheimer’s disease and stroke coexists in the mixed dementia, suggesting common pathogenic mechanisms of the two neurological diseases. Among mammalian sPLA₂s, sPLA₂-IB and sPLA₂-IIA induce neuronal apoptosis in rat cortical neurons. The excess influx of calcium into neurons via l-type voltage-dependent Ca²⁺ channels mediates the two sPLA₂-induced apoptosis. The elevated concentration of intracellular calcium activates PKC, MAPK and cytosolic PLA₂. Moreover, it is linked with the production of reactive oxygen species and apoptosis through activation of the superoxide producing enzyme NADPH oxidase. NADPH oxidase is involved in the neurotoxicity of amyloid β peptide, which impairs synaptic plasticity long before its deposition in the form of amyloid plaques of Alzheimer’s disease. In turn, reactive oxygen species from NADPH oxidase can stimulate ERK1/2 phosphorylation and activation of cPLA₂ and result in a release of arachidonic acid. sPLA₂ is up-regulated in both Alzheimer’s disease and cerebrovascular disease, suggesting the involvement of sPLA₂ in the common pathogenic mechanisms of the two diseases. Thus, our review presents evidences for pathophysiological roles of sPLA₂ in the central nervous system and neurological diseases.     

TRANSCELLULAR BIOSYNTHESIS OF CYSTEINYL LEUKOTRIENES BY KUPFFER CELL—HEPATOCYTE COOPERATION IN RAT LIVER

We previously proposed that an enzymatic cooperation between Kupffer cells and hepatocytes may play an important role in cysteinyl leukotriene (LT) production in rat liver. Anin vitrotranscellular synthesis cysteinyl LTs by a Kupffer cell—hepatocyte coculture system was characterized here. Kupffer cells alone, with A23187 stimulation, did not generate cysteinyl LTs until supplemented either with isolated hepatocytes or with LTC₄synthase and glutathione, indicating that Kupffer cells can synthesize LTA₄but not convert it into LTC₄. In contrast, hepatocytes converted the LTA₄into cysteinyl LTs and further degraded the cysteinyl LTs. Cysteinyl LT production by the Kupffer cell—hapatocyte coculture system was optimized by addition of 1–3% serum albumin to the culture and by bringing the cell—cell distance closer to less than 3μ. Tumour necrosis factor also stimulated cysteinyl LT production by the coculture system. From these results, it is expected that the Kupffer cell—hepatocyte transcellular system for cysteinyl LT production actually functionsin vivo.     

Relationship between calcium release and NADPH oxidase inhibition in human neutrophils

The aim of this study was to investigate the possible relationship between NADPH oxidase activity and changes in cytosolic Ca²⁺ in response to different agonists. Treatment of neutrophils with leukotriene B4 (LTB₄) demonstrated characteristic changes to cytoslic Ca²⁺ yielding an EC₅₀ of 4 nM. The pA₂ values for the specific LTB₄ receptor (BLT) antagonists, U-75302 and LY-255283 were 6.32 and 6.38, respectively. Similarly, neutrophils treated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and platelet activating factor (PAF) exhibited changes in cytoslic Ca²⁺ in a dose dependant manner with pD₂ values of 9.0 and 9.9, respectively. The phorbol ester PMA prevented elevations in cytosolic Ca²⁺ in response to LTB₄, FMLP and PAF with IC₅₀ values of 5.88, 1.44 and 5.71 nM, respectively. In addition, potent NADPH oxidase inhibitors apocynin and diphenyleneiodonium (DPI) inhibited FMLP mediated cytosolic Ca²⁺ release. These results demonstrate that inhibition of the NADPH oxidase suppresses cytosolic Ca²⁺ release in FMLP activated human neutrophils.     

Inhibitory effect of a peptide leukotriene antagonist ONO-1078 on LTD4- and antigen-induced thromboxane B2 production in guinea pig lungs

The effect of a peptide leukotriene receptor antagonist ONO-1078 on the production of thromboxane (Tx) B₂ induced by leukotriene (LT) D₄ and antigen challenge was examined in guinea pig lungs. LTD₄ (1–1,000 nM) induced a concentration-dependent production of TxB₂ in nonsensitized guinea pig lungs and ovalbumin challenge (0.01–100 μg/ml) produced TxB₂ and peptide leukotrienes in a concentration-dependent manner in ovalbumin-sensitized guinea pig lungs. ONO-1078 inhibited LTD₄ (100 nM)-induced TxB₂ production with the IC₅₀ value of 0.24 μM. Furthermore, ONO-1078 inhibited antigen (10 μg/ml)-induced TxB₂ production with the IC₅₀ value of 0.14 μM without effect on the production of peptide leukotrienes. These results suggest that ONO-1078 may prevent the antigen-induced production of TxB₂ through the blockade of the activation of receptors by endogenously generated peptide leukotrienes.     

Neutralization of leukotriene C4 and D4 activity by monoclonal and single-chain antibodies

Background

Cysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC₄ (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC).

Methods

We examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC₄ and LTD₄ by competing their binding to their receptors.

Results

mAbLTC and scFvLTC inhibited their binding of LTC₄ or LTD₄ to CysLT₁ receptor (CysLT₁R) and CysLT₂ receptor (CysLT₂R) overexpressed in Chinese hamster ovary cells. The induction by LTD₄ of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT₁R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC₄- and LTD₄-induced aggregation of mouse platelets expressing CysLT₂R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT₂R antagonists but not to CysLT₁R antagonists.

Conclusions

These results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT₁R and CysLT₂R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors.

General significance

mAbLTC can be used in the treatment of inflammatory diseases such as asthma.     

Discrimination between receptor- and store-operated Ca influx in human neutrophils

Ca²⁺ and Sr²⁺ entry pathways activated by pro-inflammatory agonists FMLP, LTB₄ and PAF have been compared to thapsigargin in human neutrophils. 2-APB (10 μM) increased Ca²⁺ influx and to a greater extent in agonist than in thapsigargin stimulated neutrophils. This action of 2-APB was specific to Ca²⁺ because 2-APB did not augment Sr²⁺ entry in agonist and thapsigargin stimulated neutrophils. This suggests that Ca²⁺ and Sr²⁺ entry can be used to discriminate between receptor and non-receptor (store)-operated Ca²⁺ influx. Our data show for the first time that Pyr3 whilst partially inhibiting agonist induced Ca²⁺ influx almost completely abolished Ca²⁺ influx after thapsigargin stimulation.     

Leukotrienes Are Upregulated and Associated with Human T-Lymphotropic Virus Type 1 (HTLV-1)-Associated Neuroinflammatory Disease

Leukotrienes (LTs) are lipid mediators involved in several inflammatory disorders. We investigated the LT pathway in human T-lymphotropic virus type 1 (HTLV-1) infection by evaluating LT levels in HTLV-1-infected patients classified according to the clinical status as asymptomatic carriers (HACs) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. Bioactive LTB₄ and CysLTs were both increased in the plasma and in the supernatant of peripheral blood mononuclear cell cultures of HTLV-1-infected when compared to non-infected. Interestingly, CysLT concentrations were increased in HAM/TSP patients. Also, the concentration of plasma LTB₄ and LTC₄ positively correlated with the HTLV-1 proviral load in HTLV-1-infected individuals. The gene expression levels of LT receptors were differentially modulated in CD4⁺ and CD8⁺ T cells of HTLV-1-infected patients. Analysis of the overall plasma signature of immune mediators demonstrated that LT and chemokine amounts were elevated during HTLV-1 infection. Importantly, in addition to CysLTs, IP-10 was also identified as a biomarker for HAM/TSP activity. These data suggest that LTs are likely to be associated with HTLV-1 infection and HAM/TSP development, suggesting their putative use for clinical monitoring.     

Actions of calcium influx blockers in human neutrophils support a role for receptor-operated calcium entry

The action of two potent store operated Ca²⁺ entry (SOCE) inhibitors, ML-9 and GdCl₃ on Ca²⁺ fluxes induced by the pro-inflammatory agonists FMLP, PAF, LTB₄ as well as the receptor-independent stimulus thapsigargin has not been documented in human neutrophils. In this study, ML-9 enhanced both release and subsequent Ca²⁺ influx in response to agonists whereas it enhanced Ca²⁺ release by thapsigargin, but inhibited Ca²⁺ influx. In contrast, 1 μM GdCl₃ completely inhibited Ca²⁺ influx in response to thapsigargin, but only partially blocked Ca²⁺ influx after agonist stimulation. These results strongly suggest a major role for receptor-operated Ca²⁺ influx in human neutrophils.     

Malnutrition promotes prostaglandin over leukotriene production and dysregulates eicosanoid-cytokine crosstalk in activated resident macrophages

We previously described a murine model of malnutrition that mimicked features of moderate human malnutrition, and led to increased dissemination of Leishmania donovani. In this study, we investigated the effect of malnutrition on macrophage production of cytokines, prostaglandins (PGs), and leukotrienes (LTs). Using either LPS or calcium ionophore A23187 as a stimulus, macrophages from the malnourished mice produced a 3-fold higher PG/LT ((PGE₂+6-keto-PGF_1α)/(LTB₄+cysteinyl leukotrienes)) ratio than macrophages from well-nourished mice. LPS-stimulated macrophages from the malnourished mice produced decreased levels of TNF-α, GM-CSF, and IL-10, but similar levels of IL-6 and NO compared to well-nourished mice. A complex crosstalk between the eicosanoids and cytokines in the LPS-stimulated macrophages from the malnourished mice was evident by the following: (1) high levels of PG secretion despite low levels of TNF-α; (2) supplemental IL-10 modulated the excessive PG production; (3) GM-CSF rectified the PG/LT ratio, but did not correct the abnormal cytokine profile; and (4) inhibitors of cyclooxygenase decreased the PG/LT ratio, but did not affect TNF-α. Thus, in this model of malnutrition, there is a relative increase in anti-inflammatory PGs compared to pro-inflammatory LTs, which may contribute to immunodeficiency.     

Cysteinyl leukotrienes do not mediate lipopolysaccharide-induced airway hyperresponsiveness in guinea pigs

Inhalation of bacterial lipopolysaccharide (LPS) by guinea pigs caused bronchial hyperreactivity to acetylcholine with a peak at 2 hr after exposure. Exposure to 0.01% LPS for 30 min resulted in an elevation of cysteinyl leukotrienes (cys-LTs) content in bronchoalveolar lavage fluid (BALF) which was obtained 1 hr after LPS exposure. The cys-LTs antagonist, ONO-1078 (10 mg/kg, p.o.), significantly inhibited LPS-induced bronchial hyperreactivity, but ICI-204,219 (10 mg/kg, p.o.), another cys-LT antagonist, did not. Each dose employed in the present study was sufficient to inhibit LTD₄ induced broncho-constriction in guinea pigs. In order to investigate the inhibitory mechanism of ONO-1078, the effect on the LPS-induced production of tumor necrosis factor (TNF) was examined. The amount of TNF in BALF increased significantly 2 hr after exposure to LPS. The inhalation of murine recombinant TNF-α (5 × 10⁴ u/ml) resulted in bronchial hyperreactivity in guinea pigs. ONO-1078 (10mg/kg, p.o.) inhibited the increase of LPS-induced TNF in BALF, but ICI-204,219 (10 mg/kg, p.o.) had no effect. These results suggest that TNF plays an important role in the onset of LPS-induced bronchial hyper-reactivity, and that ONO-1078 inhibits the LPS-induced airway hyperreactivity probably due to the inhibition of TNF production.     

Activation of TRPC4β by Gαi subunit increases Ca selectivity and controls neurite morphogenesis in cultured hippocampal neuron

The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca²⁺-permeable channels. TRPC channels are activated by stimulation of Gα_q-PLC-coupled receptors. Here, we report that TRPC4/TRPC5 can be activated by Gα_i. We studied the essential role of Gα_i subunits in TRPC4 activation and investigated changes in ion selectivity and pore dilation of the TRPC4 channel elicited by the Gα_i2 subunit. Activation of TRPC4 by Gα_i2 increased Ca²⁺ permeability and Ca²⁺ influx through TRPC4 channels. Co-expression of the muscarinic receptor (M2) and TRPC4 in HEK293 cells induced TRPC4-mediated Ca²⁺ influx. Moreover, both TRPC4β and the TRPC4β-Gα_i2 signaling complex induced inhibition of neurite growth and arborization in cultured hippocampal neurons. Cells treated with KN-93, a CaMKII inhibitor, prevented TRPC4- and TRPC4-Gα_i2^Q205L-mediated inhibition of neurite branching and growth. These findings indicate an essential role of Gα_i proteins in TRPC4 activation and extend our knowledge of the functional role of TRPC4 in hippocampal neurons.     

Leukotriene D4 induced calcium changes in U937 cells may utilize mechanisms additional to inositol phosphate production that are pertussis toxin insensitive but are blocked by phorbol myristate acetate

DMSO differentiated U937 cells responded to 10⁻⁶ M LTD₄, LTB₄ and FMLP with an increase in both InsP formation and [Ca²⁺]_i. FMLP caused a greater rise in InsPs than either LTD₄ or LTB₄, which were equivalent. LTD₄, however, caused a greater increase in [Ca²⁺]_i than LTB₄ (4-fold) or FMLP. The FMLP [Ca²⁺]_i and InsP responses were abolished by pertussis toxin (100 ng/ml for 4 h) but were unaffected by PMA (10⁻⁷ M for 3 min). In contrast, the LTD₄ [Ca²⁺]_i and InsP responses were reduced by only 50% by pertussis toxin, whilst PMA reduced the [Ca²⁺]_i and InsP responses to LTD₄ by 75 and 30%, respectively. These results suggest that mechanisms additional to InsP formation exist for mediating LTD₄ evoked increases in [Ca²⁺]_i.     

Antigen-Induced leukotriene relaese from rat lung

Chopped lung from inbred hyperreactive rats was challenged with antigen following active on passive sensitization and supernatants were assayed for the presence of leukotrienes (LTs) by radioimmunoassay. Dose-related increases in the release of LTC₄- and LTB₄-immunoreactive material were obtained with significantly more material being released following passive sensitization. Chromatographic analysis indicated the presence of LTB₄, LTC₄ and LTE₄. When LT release inbredred rats was compared to Sprague-Dawley or Fischer rats, the amounts released were as follows: Inbred > Sprague-Dawley > Fischer. It was concluded that the release of LTs in the three strains correlated with the degree of non-specific bronchial hyperreactivity.     

Antigen-Induced leukotriene relaese from rat lung in vitro

Chopped lung from inbred hyperreactive rats was challenged with antigen following active on passive sensitization and supernatants were assayed for the presence of leukotrienes (LTs) by radioimmunoassay. Dose-related increases in the release of LTC₄- and LTB₄-immunoreactive material were obtained with significantly more material being released following passive sensitization. Chromatographic analysis indicated the presence of LTB₄, LTC₄ and LTE₄. When LT release inbredred rats was compared to Sprague-Dawley or Fischer rats, the amounts released were as follows: Inbred > Sprague-Dawley > Fischer. It was concluded that the release of LTs in the three strains correlated with the degree of non-specific bronchial hyperreactivity.     

Group IB secretory phospholipase A2 induces neuronal cell death via apoptosis

Group IB secretory phospholipase A2 (sPLA2-IB) mediates cell proliferation, cell migration, hormone release and eicosanoid production via its receptor in peripheral tissues. In the CNS, high-affinity binding sites of sPLA2-IB have been documented. However, it remains obscure whether sPLA2-IB causes biologic or pathologic response in the CNS. To this end, we examined effects of sPLA2-IB on neuronal survival in primary cultures of rat cortical neurons. sPLA2-IB induced neuronal cell death in a concentration-dependent manner. This death was a delayed response requiring a latent time for 6 h; sPLA2-IB-induced neuronal cell death was accompanied with apoptotic blebbing, condensed chromatin, and fragmented DNA, exhibiting apoptotic features. Before cell death, sPLA2-IB liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2) from neurons. PGD2 and its metabolite, Delta12-PGJ2, exhibited neurotoxicity. Inhibitors of sPLA2 and cyclooxygenase-2 (COX-2) significantly suppressed not only AA release, but also PGD2 generation. These inhibitors significantly prevented neurons from sPLA2-IB-induced neuronal cell death. In conclusion, we demonstrate a novel biological response, apoptosis, of sPLA2-IB in the CNS. Furthermore, the present study suggests that PGD2 metabolites, especially Delta12-PGJ2, might mediate sPLA2-IB-induced apoptosis.     

Involvement of Ca<Superscript>2+</Superscript> signaling in tachykinin-mediated contractile responses in swine trachea

Summary Neuropeptide tachykinins, present within sensory nerves, have been implicated as neurotransmitters involved in nonadrenergic and noncholinergic airway muscle contraction. The signal transduction pathways of tachykinins on muscle contraction and Ca²⁺ mobilization were investigated in swine trachea. Tachykinins, substance P (SP) and neurokinin A (NKA), concentration (1 nM to 1 μM)-dependently induced contractile responses with removal of epithelium, whereas neurokinin B (NKB) did not alter the muscle tension. The SP- and NKA-evoked muscle contractions were inhibited by NK1-R antagonist L732138, but not by either NK2-R antagonist MDL29913 or NK3-R antagonist SB218795. Consistently, SP-elicited increase in [Ca²⁺]_i was abolished by NK1-R antagonist, neither by NK2-R nor NK3-R antagonists. The SP-induced muscular responses were significantly inhibited by L-type Ca²⁺ channel blocker verapamil and withdrawal of external Ca²⁺. Caffeine (10 mM) or ryanodine (50 μM) also partly suppressed the SP-induced muscle responses. Inhibition of inositol 1,4,5-trisphosphate (InsP₃) receptor with 2-APB (75 μM) potently attenuated SP-evoked Ca²⁺ mobilization and muscle contraction, which was further inhibited by 2-APB under Ca²⁺-free external solution, but not completely. Unexpectedly, simultaneous blockade of InsP₃ receptor and ryanodine receptor (RyR) by 2-APB and ryanodine enhanced SP-evoked muscle contraction and Ca²⁺ mobilization. This potentiation was virtually abolished by removal of external Ca²⁺, suggesting native Ca²⁺ channels may contribute to this phenomenon. These results demonstrate that tachykinins produce a potent muscle contraction associated with Ca²⁺ mobilization via tachykinin NK1- R-dependent activation of multiple signal transduction pathways involving Ca²⁺ influx and release of Ca²⁺ from InsP₃- and ryanodine-sensitive Ca²⁺ stores. Blockade of both InsP₃ receptor and RyR enhances the Ca²⁺ influx through native Ca²⁺ channels in plasma membrane, which is crucial to Ca²⁺ signaling in response to NK1 receptor activation.     

-alkoxyphenol leukotriene B4 receptor antagonists

A series of -alkoxyphenols containing a tetrazole acid sidechain have been prepared as antagonists of leukotriene B₄ receptors. These compounds were tested as receptor antagonists of human neutrophil and guinea pig lung membrane leukotriene B₄ receptors. Compounds in this series were found to be up to 18-fold more potent than LY255283. These results indicate that the acyl group of the 1,2,4,5 substituted hydroxyacetophenone class of LTB₄ antagonists is not critical to antagonist potency.