Activation of human inflammatory cells by secreted phospholipases A2

Secreted phospholipases A(2) (sPLA(2)s) are enzymes detected in serum and biological fluids of patients with various inflammatory, autoimmune and allergic disorders. Different isoforms of sPLA(2)s are expressed and released by human inflammatory cells, such as neutrophils, eosinophils, T cells, monocytes, macrophages and mast cells. sPLA(2)s generate arachidonic acid and lysophospholipids thus contributing to the production of bioactive lipid mediators in inflammatory cells. However, sPLA(2)s also activate human inflammatory cells by mechanisms unrelated to their enzymatic activity. Several human and non-human sPLA(2)s induce degranulation of mast cells, neutrophils and eosinophils and activate exocytosis in macrophages. In addition some, but not all, sPLA(2) isoforms promote cytokine and chemokine production from macrophages, neutrophils, eosinophils, monocytes and endothelial cells. These effects are primarily mediated by binding of sPLA(2)s to specific membrane targets (heparan sulfate proteoglycans, M-type, N-type or mannose receptors) expressed on effector cells. Thus, sPLA(2)s may play an important role in the initiation and amplification of inflammatory reactions by at least two mechanisms: production of lipid mediators and direct activation of inflammatory cells. Selective inhibitors of sPLA(2)-enzymatic activity and specific antagonists of sPLA(2) receptors are current being tested for pharmacological treatment of inflammatory and autoimmune diseases.     

Purified group X secretory phospholipase A(2) induced prominent release of arachidonic acid from human myeloid leukemia cells

Group X secretory phospholipase A(2) (sPLA(2)-X) possesses several structural features characteristic of both group IB and IIA sPLA(2)s (sPLA(2)-IB and -IIA) and is postulated to be involved in inflammatory responses owing to its restricted expression in the spleen and thymus. Here, we report the purification of human recombinant COOH-terminal His-tagged sPLA(2)-X, the preparation of its antibody, and the purification of native sPLA(2)-X. The affinity-purified sPLA(2)-X protein migrated as various molecular species of 13-18 kDa on SDS-polyacrylamide gels, and N-glycosidase F treatment caused shifts to the 13- and 14-kDa bands. NH(2)-terminal amino acid sequencing analysis revealed that the 13-kDa form is a putative mature sPLA(2)-X and the 14-kDa protein possesses a propeptide of 11 amino acid residues attached at the NH(2) termini of the mature protein. Separation with reverse-phase high performance liquid chromatography revealed that N-linked carbohydrates are not required for the enzymatic activity and pro-sPLA(2)-X has a relatively weak potency compared with the mature protein. The mature sPLA(2)-X induced the release of arachidonic acid from phosphatidylcholine more efficiently than other human sPLA(2) groups (IB, IIA, IID, and V) and elicited a prompt and marked release of arachidonic acid from human monocytic THP-1 cells compared with sPLA(2)-IB and -IIA with concomitant production of prostaglandin E(2). A prominent release of arachidonic acid was also observed in sPLA(2)-X-treated human U937 and HL60 cells. Immunohistochemical analysis of human lung preparations revealed its expression in alveolar epithelial cells. These results indicate that human sPLA(2)-X is a unique N-glycosylated sPLA(2) that releases arachidonic acid from human myeloid leukemia cells more efficiently than sPLA(2)-IB and -IIA.     

15-deoxy-Delta12,14-prostaglandin J2 inhibits IFN-inducible protein 10/CXC chemokine ligand 10 expression in human microglia: mechanisms and implications

Regulation of cytokine and chemokine expression in microglia may have implications for CNS inflammatory disorders. In this study we examined the role of the cyclopentenone PG 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) in microglial inflammatory activation in primary cultures of human fetal microglia. 15d-PGJ(2) potently inhibited the expression of microglial cytokines (IL-1, TNF-alpha, and IL-6). We found that 15d-PGJ(2) had differential effects on the expression of two alpha-chemokines; whereas the Glu-Lys-Arg (ELR)(-) chemokine IFN-inducible protein-10/CXCL10 was inhibited, the ELR(+) chemokine IL-8/CXCL8 was not inhibited. These findings were shown in primary human microglia and the human monocytic cells line THP-1 cells, using diverse cell stimuli such as bacterial endotoxin, proinflammatory cytokines (IL-1 and TNF-alpha), IFN-beta, and HIV-1. Furthermore, IL-8/CXCL8 expression was induced by 15d-PGJ(2) alone or in combination with TNF-alpha or HIV-1. Combined results from EMSA, Western blot analysis, and immunocytochemistry showed that 15d-PGJ(2) inhibited NF-kappaB, Stat1, and p38 MAPK activation in microglia. Adenoviral transduction of super-repressor IkappaBalpha, dominant negative MKK6, and dominant negative Ras demonstrated that NF-kappaB and p38 MAPK were involved in LPS-induced IFN-inducible protein 10/CXCL10 production. Interestingly, although LPS-induced IL-8/CXCL8 was dependent on NF-kappaB, the baseline or 15d-PGJ(2)-mediated IL-8/CXCL8 production was NF-kappaB independent. Our results demonstrate that 15d-PGJ(2) has opposing effects on the expression of two alpha-chemokines. These data may have implications for CNS inflammatory diseases.     

Presence of the M-type sPLA(2) receptor on neutrophils and its role in elastase release and adhesion

Secretory phospholipase A(2) (sPLA(2)) produces lipids that stimulate polymorphonuclear neutrophils (PMNs). With the discovery of sPLA(2) receptors (sPLA(2)-R), we hypothesize that sPLA(2) stimulates PMNs through a receptor. Scatchard analysis was used to determine the presence of a sPLA(2) ligand. Lysates were probed with an antibody to the M-type sPLA(2)-R, and the immunoreactivity was localized. PMNs were treated with active and inactive (+EGTA) sPLA(2) (1-100 units of enzyme activity/ml, types IA, IB, and IIA), and elastase release and PMN adhesion were measured. PMNs incubated with inactive, FITC-linked sPLA(2)-IB, but not sPLA(2)-IA, demonstrated the presence of a sPLA(2)-R with saturation at 2.77 fM and a K(d) of 167 pM. sPLA(2)-R immunoreactivity was present at 185 kDa and localized to the membrane. Inactive sPLA(2)-IB activated p38 MAPK, and p38 MAPK inhibition attenuated elastase release. Active sPLA(2)-IA caused elastase release, but inactive type IA did not. sPLA(2)-IB stimulated elastase release independent of activity; inactive sPLA(2)-IIA partially stimulated PMNs. sPLA(2)-IB and sPLA(2)-IIA caused PMN adhesion. We conclude that PMNs contain a membrane M-type sPLA(2)-R that activates p38 MAPK.     

Impact of CFTR DeltaF508 mutation on prostaglandin E2 production and type IIA phospholipase A2 expression by pulmonary epithelial cells

Cystic fibrosis (CF) is characterized by an exacerbated inflammatory pulmonary response with excessive production of inflammatory mediators. We investigated here the impact of cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction on prostaglandin E2 (PGE2) production and type IIA secreted phospholipase A2 (sPLA2-IIA) expression. We show that both resting and LPS-stimulated human respiratory epithelial cell line bearing DeltaF508 mutation on CFTR (CF cells) released more PGE2 than control cell line. This was accompanied by enhanced expression and activity of cyclooxygenase-2 in CF cells. PGE2 release was attenuated after experimentally induced retrafficking of the DeltaF508-CFTR at the plasma membrane. sPLA2-IIA expression occurred at higher levels in CF cells than in control cells and was enhanced by LPS and PGE2. Suppression of PGE2 synthesis by aspirin led to an inhibition of LPS-induced sPLA2-IIA expression. Higher activation of NF-kappaB was observed in CF cells compared with control cells and was enhanced by LPS. However, addition of PGE2 or aspirin had no effect on NF-kappaB activation. LPS-induced sPLA2-IIA expression was reduced by an NF-kappaB inhibitor. We suggest that the lack of the CFTR in the plasma membrane results in a PGE2 overproduction and an enhanced sPLA2-IIA expression. This expression is upregulated by NF-kappaB and amplified by PGE2 via a unidentified signaling pathway.     

Viral IL-6 blocks neutrophil infiltration during acute inflammation

Pathologies arising as a consequence of human herpesvirus-8 (HHV8) infections are closely associated with the autocrine activity of a HHV8 encoded IL-6 (vIL-6), which promotes proliferation of infected cells and their resistance to apoptosis. In this present report, studies show that vIL-6 may also be important in influencing the host's immunological response to secondary infections. Using peritoneal inflammation as a model of acute bacterial infection, vIL-6 was found to specifically block neutrophil recruitment in vivo through regulation of inflammatory chemokine expression. This response was substantiated in vitro where activation of STAT3 in human peritoneal mesothelial cells by vIL-6 was associated with enhanced CCL2 release. Although vIL-6 did not effect CXCL8 production, IL-1beta-induced secretion of this neutrophil-activating chemokine was significantly suppressed by vIL-6. These data suggest that vIL-6 has the capacity to suppress innate immune responses and thereby influence the outcome of opportunistic infections in HHV8-associated disease.     

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.     

Secreted phospholipases A2 induce the expression of chemokines in microvascular endothelium

Acute respiratory distress syndrome (ARDS) is characterized by alterations in microvascular permeability. In ARDS secreted phospholipase A(2) (sPLA(2)) IB and IIA are found to be highly upregulated. In this study, we therefore investigated the influence of exogenously added sPLA(2)-IB and sPLA(2)-IIA on the production of chemokines and adhesion molecules in lung microvascular endothelial cells (LMVEC). Treatment of LMVEC with sPLA(2)s resulted in a significant increase in the production of chemokines and adhesion molecules due to an increased expression of their mRNA and in an enhanced release of oleic acid. The upregulation of chemokines and adhesion molecules by LPS was stronger in the presence of sPLA(2). Activation of NF-kappaB occurred upon stimulation with sPLA(2). Moreover the MAPkinase pERK seems to be involved since a specific pERK inhibitor, e.g., U0126, but not a p38Kinase inhibitor, e.g., SB203580 prevented sPLA(2)-induced chemokine upregulation. Our data therefore suggest that LMVEC are a highly sensitive target for the direct action of extracellular sPLA(2)s.     

Activation profile of CXCL8-stimulated neutrophils and aging

Neutrophils are pivotal effector cells of innate immunity representing the first line of defense against aggression. They are the first cells to arrive at the site of the aggression, where they can directly eliminate the invading microorganisms. Their activation and recruitment into peripheral tissues is indispensable for host defense. With aging, there are alterations of the receptor by driven functions of human neutrophils as a decrease in the functional changes in signaling elicited by specific receptors, as CXCR1. We investigated the activation of neutrophils from elderly after the cells were cultivated with CXCL8. Although, CXCL8 induced elastase (ELA) secretion, data showed neither myeloperoxidase (MPO) activity nor production of IL-6, IL-10, GM-CSF by neutrophils from elderly compared with young individuals. On the other hand, in the presence of only LPS or LPS associated with CXCL8 neutrophils from elderly individuals, there were significant levels of IL-6, IL-10, GM-CSF but not MPO. These results indicate that neutrophils from elderly do not respond to CXCL8 stimulus, but they are activated by LPS to produce cytokines. However, MPO activity from elderly individuals was not different in the presence or absence of LPS and CXCL8.     

Outer membrane protein A from Klebsiella pneumoniae activates bronchial epithelial cells: implication in neutrophil recruitment

Aside from its mechanical barrier function, bronchial epithelium plays an important role both in the host defense and in the pathogenesis of inflammatory airway disorders. To investigate its role in lung defense, the effect of a bacterial cell wall protein, the outer membrane protein A from Klebsiella pneumoniae (kpOmpA) on bronchial epithelial cells (BEC) was evaluated on adhesion molecule expression and cytokine production. Moreover, the potential implication of this mechanism in kpOmpA-induced lung inflammation was also determined. Our in vitro studies demonstrated that kpOmpA strongly bound to BEAS-2B cells, a human BEC line, and to BEC primary cultures, resulting in NF-kappaB signaling pathway activation. Exposure to kpOmpA increased ICAM-1 mRNA and cell surface expression, as well as the secretion of IL-6, CXC chemokine ligand (CXCL)1, CXCL8, C-C chemokine ligand 2, CXCL10 by BEAS-2B cells, and BEC primary cultures (p < 0.005). We analyzed in vivo the consequences of intratracheal injection of kpOmpA to BALB/c mice. In kpOmpA-treated mice, a transient neutrophilia (with a maximum at 24 h) was observed in bronchoalveolar lavage and lung sections. In vivo kpOmpA priming induced bronchial epithelium activation as evaluated by ICAM-1 and CXCL1 expression, associated with the secretion of CXCL1 and CXCL5 in bronchoalveolar lavage fluids. In the lung, an increased level of the IL-6, CXCL1, CXCL5, CXCL10 mRNA was observed with a maximum at 6 h. These data showed that kpOmpA is involved in host defense mechanism by its ability to activate not only APC but also BEC, resulting in a lung neutrophilia.     

Monocyte-astrocyte networks and the regulation of chemokine secretion in neurocysticercosis

Neurocysticercosis, caused by infection with larval Taenia solium, is a major cause of epilepsy worldwide. Larval degeneration, which is symptomatic, results in inflammatory cell influx. Astrocytes, the most abundant cell type and major cytokine-producing cell within the CNS, may be important in orchestrating inflammatory responses after larval degeneration. We investigated the effects of direct stimulation and of conditioned medium from T. solium larval Ag (TsAg)-stimulated monocytes (CoMTsAg) on neutrophil and astrocyte chemokine release. CoMTsAg, but not control conditioned medium, stimulated astrocyte CCL2/MCP-1 (161.5 +/- 16 ng/ml), CXCL8/IL-8 (416 +/- 6.2 ng/ml), and CXCL10/IFN-gamma-inducible protein (9.07 +/- 0.6 ng/ml) secretion after 24 h, whereas direct astrocyte or neutrophil stimulation with TsAg had no effect. There was rapid accumulation of CCL2 and CXCL8 mRNA within 1 h, with somewhat delayed expression of CXCL10 mRNA initially detected 8 h poststimulation. Neutralizing anti-TNF-alpha inhibited CoMTsAg-induced CCL2 mRNA accumulation by up to 99%, causing total abolition of CXCL10 and up to 77% reduction in CXCL8 mRNA. CoMTsAg induced maximal nuclear binding of NF-kappaB p65 and p50 by 1 h, with IkappaBalpha and IkappaBbeta decay within 15 min. In addition, CoMTsAg induced transient nuclear binding of AP-1, which peaked 4 h poststimulation. In NF-kappaB blocking experiments using pyrrolidine dithiocarbamate, CoMTsAg-induced CCL2 secretion was reduced by up to 80% (p = 0.0006), whereas CXCL8 was inhibited by up to 75% (p = 0.0003). In summary, the data show that astrocytes are an important source of chemokines following larval Ag stimulation. Such chemokine secretion is NF-kappaB dependent, likely to involve AP-1, and is regulated in a paracrine loop by monocyte-derived TNF-alpha.