Labour is associated with increased expression of type-IIA secretory phospholipase A2 but not type-IV cytosolic phospholipase A2 in human myometrium

Human labour is associated with increased prostaglandin synthesis within the uterus. The aim of this study was to examine the expression of the type-IV cytosolic phospholipase A2 (cPLA2-IV) and the type IIA secretory phospholipase A2 (sPLA2-IIA) in myometrium in association with labour onset at term and preterm deliveries. These enzymes are important for the release of the prostaglandin precursor, arachidonic acid, from phospholipid membrane stores. RT-PCR was used to determine differences in gene expression between non-labour and labour groups. Expression of sPLA2-IIA in human myometrium was significantly increased with pregnancy, and with labour, both at term and preterm. Expression of cPLA2-IV in myometrium was not significantly altered with respect to pregnancy or labour. Immunohistochemical analysis demonstrated differences in the spatial localization of cPLA2-IV and sPLA2-IIA protein in upper and lower segment myometrium. cPLA2-IV was predominantly in vascular endothelial cells, while sPLA2-IIA was observed in vascular, endothelial and smooth muscle cells. In addition, sPLA2-IIA showed a distinct nuclear or perinuclear localization in myometrial smooth muscle cells of the lower segment. We postulate that the increased expression of sPLA2-IIA rather than cPLA2-IV in the myometrium may play a role in the onset and/or maintenance of human parturition.     

Crucial role of group IIA phospholipase A2 in pancreatitis-associated adrenal injury in acute necrotizing pancreatitis

This study investigated the mechanistic role of group IIA phospholipase A2 (sPLA2-IIA) in the process of pancreatitis-associated adrenal injury in acute necrotizing pancreatitis.     

Lack of macrophage migration inhibitory factor in mice does not affect hallmarks of the inflammatory/immune response during the first week after stroke

Background

Activation of glial cells, including astrocytes and microglia, has been implicated in the inflammatory responses underlying brain injury and neurodegenerative diseases including Alzheimer's and Parkinson's diseases. Although cultured astrocytes and microglia are capable of responding to pro-inflammatory cytokines and lipopolysaccharide (LPS) in the induction and release of inflammatory factors, no detailed analysis has been carried out to compare the induction of iNOS and sPLA2-IIA. In this study, we investigated the effects of cytokines (TNF-alpha, IL-1beta, and IFN-gamma) and LPS + IFN-gamma to induce temporal changes in cell morphology and induction of p-ERK1/2, iNOS and sPLA₂-IIA expression in immortalized rat (HAPI) and mouse (BV-2) microglial cells, immortalized rat astrocytes (DITNC), and primary microglia and astrocytes.

Methods/Results

Cytokines (TNF-alpha, IL-1beta, and IFN-gamma) and LPS + IFN-gamma induced a time-dependent increase in fine processes (filopodia) in microglial cells but not in astrocytes. Filopodia production was attributed to IFN-gamma and was dependent on ERK1/2 activation. Cytokines induced an early (15 min) and a delayed phase (1 ~ 4 h) increase in p-ERK1/2 expression in microglial cells, and the delayed phase increase corresponded to the increase in filopodia production. In general, microglial cells are more active in responding to cytokines and LPS than astrocytes in the induction of NO. Although IFN-gamma and LPS could individually induce NO, additive production was observed when IFN-gamma was added together with LPS. On the other hand, while TNF-alpha, IL-1beta, and LPS could individually induce sPLA₂-IIA mRNA and protein expression, this induction process does not require IFN-gamma. Interestingly, neither rat immortalized nor primary microglial cells were capable of responding to cytokines and LPS in the induction of sPLA2-IIA expression.

Conclusion

These results demonstrated the utility of BV-2 and HAPI cells as models for investigation on cytokine and LPS induction of iNOS, and DITNC astrocytes for induction of sPLA2-IIA. In addition, results further demonstrated that cytokine-induced sPLA2-IIA is attributed mainly to astrocytes and not microglial cells.     

The expression of genes encoding for COX-2, MPO, iNOS, and sPLA2-IIA in patients with recurrent depressive disorder

BackgroundThere is evidence that inflammation, oxidative and nitrosative stress (IO&;NS) play a role in the pathophysiology of depression. There are also data indicating altered inflammatory gene expression in depressive disorder and that genetic variants of IO&;NS genes are associated with increased risk of the disease in question. The aim of this study was to explore mRNA expression of four IO&;NS genes PTGS2, MPO, NOS2A, and PLA2G2A coding respectively: cyclooxygenase-2 (COX-2), myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS) and secretory phospholipase A2 type IIA (sPLA2-IIA).MethodExpression of the mRNA was determined using quantitative real-time PCR, in peripheral blood cells of patients with recurrent depressive disorder (rDD) and normal controls.ResultsThe mRNA expressions of the genes encoding for COX-2, MPO, iNOS and sPLA2-IIA were significantly increased in the peripheral blood cells of depressed patients versus controls.LimitationsPatients were treated with antidepressants.ConclusionOur results indicate and may confirm the role of peripheral IO&;NS pathways in the pathophysiology of depression. The results represent a promising way to investigate biological markers of depression.     

Macrophage mannose receptor in chronic sinus disease

BACKGROUND: The role of infectious agents in the onset and maintenance of chronic sinus disease is still not fully understood. Macrophage mannose receptor (MMR), an innate pattern recognizing receptor, capable of phagocytosis of invaders and signal transduction for proinflammatory mechanisms, might be of importance in immune interactions in chronic sinus disease. OBJECTIVE: We examined the MMR in sinonasal airway mucosa to evaluate its possible role in chronic rhinosinusitis (CS) and nasal polyposis (NPs). METHODS: Surgical samples from patients with sinonasal disease were investigated with real-time RT-PCR for quantification of MMR mRNA expression, and the presence and location of MMR-positive cells was analysed by immunohistochemistry. RESULTS: Quantification of MMR mRNA showed a statistically significant higher expression in NPs compared to CS without NP and controls. Immunohistochemistry revealed expression of MMR in all tissue samples; however, in NP we found an enhanced positive cellular staining including cell aggregates. CONCLUSIONS: We could demonstrate for the first time that the expression of MMR is significantly upregulated in NP compared to patients with CS without NP or turbinate tissue of controls. Macrophages expressing MMR, accumulated in cell aggregates in NPs, play a possible key role in pathogen-macrophage interaction in NP disease.     

Prostaglandin, leukotriene, and lipoxin balance in chronic rhinosinusitis with and without nasal polyposis

BACKGROUND: Upper airway diseases and especially the aspirin hypersensitivity syndrome have been linked to changes in the arachidonic acid cascade; however, the specificity of these changes and their relation to inflammatory reactions in these diseases still remain controversial. OBJECTIVE: We aimed to study the tissue eicosanoid production in 3 subgroups of patients with chronic rhinosinusitis (CRS) and control subjects and to correlate it with the severity of inflammation and clinical manifestation of aspirin sensitivity. METHODS: Samples were prepared from sinonasal tissue of patients with CRS with (CRS-NP group, n = 13) and without nasal polyposis (CRS group, n = 11), sinonasal tissue of patients with nasal polyposis and aspirin sensitivity (CRS-ASNP group, n = 13), and normal nasal mucosa from healthy subjects (NM group, n = 8). Real-time PCR was applied for mRNA quantification of COX-2, 5-lipoxygenase, leukotriene C 4 synthase, and 15-lipoxygenase. Enzyme immunoassays were used to measure IL-5, eosinophil cationic protein, and eicosanoid (leukotriene [LT] C 4 , LTD 4 , and LTE 4 ; lipoxin A 4 ; and prostaglandin E 2 [PGE 2 ]) concentrations. RESULTS: COX-2 mRNA and PGE 2 concentrations were similar in the CRS and NM groups but significantly decreased in nasal polyp tissue, especially in the CRS-ASNP group. LTC 4 synthase, 5-lipoxygenase mRNA, LTC 4 , LTD 4 , and LTE 4 concentrations increased with disease severity among the patient groups. 15-Lipoxygenase and lipoxin A 4 concentrations were increased in all CRS groups compared with in the NM group but were significantly downregulated in the CRS-ASNP group when compared with the CRS-NP group. IL-5 and eosinophil cationic protein were increased in both groups of nasal polyp tissue compared with in the NM and CRS groups and correlated directly with LTC 4 , LTD 4 , and LTE 4 concentrations and inversely with PGE 2 concentrations. CONCLUSION: Changes of tissue eicosanoid metabolism do occur in CRS, even in the absence of clinical aspirin sensitivity, and these changes appear to be related to the severity of eosinophilic inflammation.     

Secretory phospholipases A2 induce cytokine release from blood and synovial fluid monocytes.

Secretory phospholipases A2 (sPLA2) are released in the blood of patients with various inflammatory diseases and exert proinflammatory activities by releasing arachidonic acid (AA), the precursor of eicosanoids. We examined the ability of four sPLA2 to activate blood and synovial fluid monocytes in vitro. Monocytes were purified from blood of healthy donors or from synovial fluid of patients with rheumatoid arthritis by negative immunoselection and by adherence to plastic dishes, respectively. The cells were incubated with group IA, IB, IIA and III sPLA2 and the release of TNF-alpha, IL-6 and IL-12 was determined by ELISA. Group IA, IB and IIA sPLA2 induced a concentration-dependent release of TNF-alpha and IL-6 from blood monocytes. These sPLA2 activated IL-12 production only in monocytes preincubated with IFN-gamma. Group IA and IIA sPLA2 also induced TNF-alpha and IL-6 release from synovial fluid monocytes. TNF-alpha and IL-6 release paralleled an increase in their mRNA expression and was independent from the capacity of sPLA2 to mobilize AA. These results indicate that sPLA2 stimulate cytokine release from blood and synovial fluid monocytes by a mechanism at least partially unrelated to their enzymatic activity. This effect may concur with the generation of AA in the proinflammatory activity of sPLA2 released during inflammatory diseases.     

Tagging SNP haplotype analysis of the secretory PLA2-V gene, PLA2G5, shows strong association with LDL and oxLDL levels, suggesting functional distinction from sPLA2-IIA: results from the UDACS study

Animal and human studies suggest that both secretory PLA2 (sPLA2)-V and sPLA2-IIA (encoded, respectively, by the neighbouring PLA2G5 and PLA2G2A genes) contribute to atherogenesis. Elevated plasma sPLA2-IIA predicts coronary heart disease (CHD) risk, but no mass assay for sPLA2-V is available. We previously reported that tagging single nucleotide polymorphism (tSNP) haplotypes of PLA2G2A are strongly associated with sPLA2-IIA mass, but not lipid levels. Here, we use tSNPs of the sPLA2-V gene to investigate the association of PLA2G5 with CHD risk markers. Seven PLA2G5 tSNPs genotypes, explaining >92% of the locus genetic variability, were determined in 519 patients with Type II diabetes (in whom PLA2G2A tSNP data was available), and defined seven common haplotypes (frequencies >5%). PLA2G5 and PLA2G2A tSNPs showed linkage disequilibrium (LD). Compared to the common PLA2G5 haplotype, H1 (frequency 34.9%), haplotypes H2-7 were associated with overall higher plasma LDL (P < 0.00004) and total cholesterol (P < 0.00003) levels yet lower oxLDL/LDL (P = 0.006) and sPLA2-IIA mass (P = 0.04), probably reflecting LD with PLA2G2A. Intronic tSNP (rs11573248), unlikely itself to be functional, distinguished H1 from LDL-raising haplotypes and may mark a functional site. In conclusion, PLA2G5 tSNP haplotypes demonstrate an association with total and LDL cholesterol and oxLDL/LDL, not seen with PLA2G2A, thus confirming distinct functional roles for these two sPLA2s.     

Specific differential expression of phospholipase A2 subtypes in rat cerebellum

Phospholipase A2 (PLA2) is a family of enzymes playing diverse roles in lipid signaling in neurons and glia cells. In this study, we examined the expression of subtypes of PLA2 in the cerebellum using immunolabeling and in situ hybridization methods. Two Ca2+-dependent cytosolic subtypes (cPLA2alpha and cPLA2beta), one Ca2+-independent cytosolic subtype (iPLA2), and two secretory subtypes (sPLA2IIA and sPLA2V) were detected in the cerebellum. cPLA2alpha is present in somata and dendrites of Purkinje cells, while sPLA2IIA is associated with the endoplasmic reticulum in perinuclear regions of Purkinje cell somata. iPLA2 is present in granule cells, stellate cells and also in the nucleus of Purkinje cells. In addition, cPLA2beta is localized in granule cells, and sPLA2V in Bergmann glia cells. These results provide an important basis for identifying functional roles of PLA2s in the cerebellum.     

Secreted phospholipase A2-IIA-induced a phenotype of activated microglia in BV-2 cells requires epidermal growth factor receptor transactivation and proHB-EGF shedding

ABSTRACT: BACKGROUND: Activation of microglia, the primary component of the innate immune response in the brain, is a hallmark of neuroinflammation in neurodegenerative disorders, including Alzheimer's disease (AD) and other pathological conditions such as stroke or CNS infection. In response to a variety of insults, microglial cells produce high levels of inflammatory cytokines that are often involved in neuronal injury, and play an important role in the recognition, engulfment, and clearance of apoptotic cells and/or invading microbes. Secreted phospholipase A2-IIA (sPLA2-IIA), an enzyme that interacts with cells involved in the systemic immune/inflammatory response, has been found up-regulated in the cerebrospinal fluid and brain of AD patients. However, despite several approaches, its functions in mediating CNS inflammation remain unknown. In the present study, the role of sPLA2-IIA was examined by investigating its direct effects on microglial cells. METHODS: Primary and immortalized microglial cells were stimulated by sPLA2-IIA in order to characterize the cytokine-like actions of the phospholipase. The hallmarks of activated microglia analyzed include: mitogenic response, phagocytic capabilities and induction of inflammatory mediators. In addition, we studied several of the potential molecular mechanisms involved in those events. RESULTS: The direct exposure of microglial cells to sPLA2-IIA stimulated, in a time- and dose-dependent manner, their phagocytic and proliferative capabilities. sPLA2-IIA also triggered the synthesis of the inflammatory proteins COX-2 and TNFalpha. In addition, EGFR phosphorylation and shedding of the membrane-anchored heparin-binding EGF-like growth factor (pro-HB-EGF) ectodomain, as well as a rapid activation/phosphorylation of the classical survival proteins ERK, P70S6K and rS6 were induced upon sPLA2-IIA treatment. We further demonstrated that the presence of an EGFR inhibitor (AG1478), a matrix metalloproteinase inhibitor (GM6001), an ADAM inhibitor (TAPI-1), and a HB-EGF neutralizing antibody abrogated the phenotype of activated microglia induced by the sPLA2-IIA. CONCLUSION: These results support the hypothesis that sPLA2-IIA may act as a potent modulator of microglial functions through its ability to induce EGFR transactivation and HB-EGF release. Accordingly, pharmacological modulation of EGFR might be a useful tool for treating neuroinflammatory diseases characterized by sPLA2-IIA accumulation.     

Chronic rhinosinusitis with and without nasal polyps is associated with decreased expression of glucocorticoid-induced leucine zipper

Background Chronic rhinosinusitis without nasal polyps (CRSsNP) and with nasal polyps (CRSwNP) is characterized by persistent inflammation of sinonasal mucosa. Glucocorticoid-induced leucine zipper (GILZ) is a recently described anti-inflammatory mediator.
Objective Here we analysed the expression of GILZ in CRSsNP and CRSwNP, its association with response to surgery, and its cytokine-driven expression regulation in the upper airways.
Methods The messenger RNA (mRNA) and protein expression of GILZ in 33 CRSsNP, 32 CRSwNP, and 11 control samples was assessed by means of a quantitative RT-PCR and immunohistochemistry, respectively. Nasal explant culture was used to investigate the effect of IFN-γ, IL-4, IL-13, IL-1β, and TNF-α on GILZ mRNA expression in normal sinonasal mucosa.
Results The GILZ mRNA and protein expression was significantly suppressed in both CRSsNP and CRSwNP patients compared with controls. No significant difference in GILZ expression was found between CRSsNP and CRSwNP patients. Comparing patients responsive and patients recalcitrant to surgery, a significant further decrease of GILZ expression was found in recalcitrant patients both in the CRSsNP and in the CRSwNP group. IL-1β, TNF-α, IL-4, and IL-13 reduced, whereas IFN-γ enhanced GILZ mRNA levels in the sinonasal mucosa.
Conclusion Down-regulated expression of GILZ may contribute to the pathogenesis of CRSsNP and CRSwNP and associate with response to surgery. GILZ expression in the upper airways can be regulated differentially by different cytokines.     

Cyclooxygenase 1 and cyclooxygenase 2 expression is abnormally regulated in human nasal polyps

BACKGROUND: There is evidence that impairment of prostanoid metabolism might be involved in the pathogenesis of nasal polyps (NPs). Prostanoids are synthesized by 2 cyclooxygenase (Cox) enzymes, one constitutive (Cox-1) and another inducible (Cox-2). OBJECTIVE: The aim of these studies was to investigate Cox-1 and Cox-2 regulation in NPs of aspirin-tolerant human patients compared with that seen in nasal mucosa (NM). METHODS: Cultured explants from human NPs and healthy mucosa from patients undergoing polypectomy and corrective nasal surgery, respectively, were examined for Cox-1 and Cox-2 expression by means of semiquantitative competitive PCR and Western blotting. RESULTS: Cox-1 mRNA was spontaneously upregulated in cultured NM but not in NPs. A spontaneous but delayed upregulation of Cox-2 mRNA was found in NPs (24 hours) compared with that seen in NM (6 hours). After cytokine stimulation (IFN-gamma, IL-1beta, and TNF-alpha), the induction of Cox-2 mRNA and protein was also faster in NM (1 hour) than in NPs (4 hours). CONCLUSION: These data showing an abnormal regulation of Cox-1 and Cox-2 in NPs from aspirin-tolerant patients reinforce the concept that prostanoid metabolism might be important in the pathogenesis of inflammatory nasal diseases and suggest a potential role for this alteration in the formation of NPs.     

Glandular gene expression of sinus mucosa in chronic rhinosinusitis with and without cystic fibrosis

Secretory cells in submucosal glands (SMGs) secrete antibacterial proteins and mucin glycoproteins into the apical lumen of the respiratory tract, and these are critical for innate immune mucosal integrity. Glandular hyperplasia is manifested in diseases with obstructive respiratory pathologies associated with mucous hypersecretion, and is predominant in the sinus mucosa of patients with chronic rhinosinusitis (CRS), cystic fibrosis (CF), and clinical symptoms of CRS. To gain insights into the molecular basis of SMG hyperplasia in CRS, gene expression microarray analyses were performed to identify the differences in global and specific gene expression in the sinus mucosa of control, CRS, and CRS/CF patients. A marked up-regulation of 11 glandular-associated genes in CRS and CRS/CF sinus mucosa was evident. The RNA and protein expressions of the four most highly up-regulated genes (DSG3, KRT14, PTHLH, and OTX2) were evaluated. An increased expression of DSG3, KRT14, and PTHLH was demonstrated at the mRNA and protein levels in both CRS and CRS/CF sinus mucosa, whereas the increased expression of OTX2 was evident only for CRS/CF sinus mucosa, implicating OTX2 as a CF-specific gene. Immunofluorescence analysis localized DSG3, PTHLH, and OTX2 to serous cells, and KRT14 to myoepithelial cells, in SMGs. Because glandular hyperplasia is a central histologic feature of CRS, the identification of overexpressed glandular genes in the sinus mucosa lays the groundwork for future studies of glandular hyperplasia, and may ultimately lead to the development of novel treatments for mucous hypersecretion in patients with CRS.     

Expression of group IIA secretory phospholipase A2 is elevated in prostatic intraepithelial neoplasia and adenocarcinoma

Phospholipase A2 (PLA2) enzymes release arachidonic acid from cellular phospholipids in a variety of mammalian tissues, including prostate. Group IIa secretory PLA2 (sPLA2) can generate arachidonate from cellular phospholipids. We examined the group IIa sPLA2 expression in benign prostatic tissues, prostatic intraepithelial neoplasia (PIN), and adenocarcinoma to determine whether sPLA2 expression is altered in the carcinogenesis of human prostatic cancer. Thirty-three of 74 total cases (45%) of benign prostatic tissue showed positive immunohistochemical staining for group IIA sPLA2, whereas 63 of 69 total cases (91%) of high-grade PINs and 70 of 78 total cases (90%) of adenocarcinomas gave positive results. Four of 10 cases of low-grade PIN showed positive immunoreactivity for sPLA2. The number of cells staining for sPLA2 was significantly less in benign epithelium (4%) and low-grade PIN (4%) compared to high-grade PIN (40%) or adenocarcinoma (38%) (P < 0.001). There was no significant difference between high-grade PIN and adenocarcinoma in the number of cells staining positively for sPLA2. The intensity of sPLA2 immunoreactivity was also different among benign prostatic tissue, low-grade PIN, high-grade PIN, and prostatic adenocarcinoma specimens. The malignant cells demonstrated more intense immunohistochemical staining (moderate to strong staining in 81% and 69% cases for high-grade PIN and adenocarcinoma, respectively) than benign glands (moderate staining in 11% of cases). No strong staining was observed in benign glands or low-grade PIN. Our data are consistent with the contention that group IIA sPLA2 expression is elevated in neoplastic prostatic tissue and support the hypothesis that dysregulation of sPLA2 may play a role in prostatic carcinogenesis.     

Myometrial prostaglandin E2 synthetic enzyme mRNA expression: spatial and temporal variations with pregnancy and labour

We have investigated the hypothesis that the expression of the enzymes involved in PGE(2) synthesis in the human uterus is co-ordinated. We have studied (i) the mRNA expression of the enzymes involved in PGE(2) synthesis [phospholipases (cPLA(2) and sPLA(2)), prostaglandin H synthase (PGHS)-2 and PG E synthases (PGES-1 and -2)] and their relationship to the expression of inflammatory cytokines in samples of myometrium obtained from pregnant women undergoing caesarean section (LSCS) either before or after the onset of labour at or before term; and (ii) the effect of IL-1beta, IL-6, TNF-alpha, PGE(2) and stretch on PGE(2) enzyme mRNA expression. We found that cPLA(2), sPLA(2) and PGHS-2 mRNA expression were greater in labour samples; cPLA(2), sPLA(2), PGHS-2, PGES-1 and -2 mRNA expression were greater in lower- than upper-segment samples; and there was no effect of gestational age. PGHS-2 mRNA levels correlated with those of PGES-1, cPLA(2), IL-1beta and IL-8; PGES-1 mRNA levels correlated with those of IL-1beta, IL-8 and cPLA(2). In primary cultures of uterine myocytes, cPLA(2) mRNA expression was increased by IL-1beta and IL-6; PGHS-2 mRNA expression was increased by IL-1beta, PGE(2) and stretch; and PGES-1 mRNA expression was increased by IL-1beta only. These data show that labour is associated with increased expression of the enzymes involved in PGE(2) synthesis and their expression is greater in the lower uterine segment. The presence of associations between the levels of PGE(2) enzyme mRNA expression and the effects of IL-1beta suggest that their expression is co-ordinated and that IL-1beta is the responsible factor.     

The expression and distribution of group IIA phospholipase A2 in human colorectal tumours

Secretory phospholipase A2 group IIA (IIA PLA2) is a protein shown to be increased in various human malignancies. The expression profile of this protein, however, is controversial in colorectal carcinoma. The aim of this study was to examine the distribution and expression of IIA PLA2 protein in benign, premalignant and malignant colorectal tumours as well as in peritumoural mucosa. Seven hyperplastic polyps, 24 adenomas and 83 colorectal carcinomas were stained with immunohistochemistry (IHC) for IIA PLA2. Four hyperplastic polyps, 12 adenomas and nine carcinomas were also evaluated for the sites of IIA PLA2 expression using mRNA in situ hybridisation (ISH). There was no immunoreactivity for IIA PLA2 in hyperplastic polyps. A total of 79% of adenomas and 31% of carcinomas showed IIA PLA2-immunopositive tumour cells in IHC, and the expression was localised to epithelial cells with ISH. In carcinomas, IIA PLA2-immunopositive apoptotic cells and necrosis were also found. The epithelial cells in the peritumoural mucosa showed immunopositivity for IIA PLA2 in 96% of cases, with considerably stronger intensity adjacent to carcinoma than in the more distal mucosa. Moreover, IIA PLA2-immunopositive malignant epithelial cells were found in 44% of cases in the invasive front of carcinomas. Our results suggest that the IIA PLA2 protein content is dramatically decreased in malignant colorectal tumours as compared with adenomas. The protein is also found in the apoptotic cells, necrosis, peritumoural mucosa and in the invasive front of carcinomas.