Inhibition of the activity of pro-inflammatory secretory phospholipase A2 by acute phase proteins

Pro-Inflammatory non-pancreatic phospholipase A2 (sPLA2) is markedly over-expressed in acute systemic and chronic local inflammatory processes. Since in acute phase reaction sPLA2 is often over-expressed simultaneously with acute phase proteins (APP), it is important to determine whether APP interacts with sPLA2. We tested ten APPs for interaction with sPLA2 using as a substrate multilamellar Hposomes composed either of PC:Lyso PC or PE:Lyso PE. Using PC:Lyso PC substrate, CRP, lactoferrin and SAP were found to inhibit sPLA2 activity with an IC50 of 25 μg/ml, 7.5 μg/ml and 50 μg/ml, respectively, corresponding to 0.21 μM, 0.1 μM and 0.21 μM respectively. Using PE:Lyso PE substrate only SAP was inhibitory, with an IC50 of 10 μg/ml (0.04 μM). Phosphorylcholine abolished the inhibitory activity of CRP but not of SAP or lactoferrin. Addition of phosphorylethanolamine or of excess calcium had no effect on the inhibitory activity of APP. Limulin, lysozyme, transferrin, β2-microglobulin, α2-macroglobulin, human and bovine albumins had no effect on sPLA2 activity. Therefore neither the structure of pentraxins, or ironbinding, bacteriostatic property or amyloidogenic property preclude whether APP modulates sPLA2 activity. Inhibition of pro-inflammatory sPLA2 by APP may be one of the protective mechanisms of the acute phase reaction

Sequential release of TNFα and phospholipase A2 in a rat model of LPS-induced pleurisy

The levels of extracellular phospholipase A2 (sPLA2) and TNFα, and cell accumulation were measured in the pleural washings obtained at different times following the induction of Escherichia coli lipopolysaccharide (LPS, 100 μg/cavity) pleurisy in rats. TNFα peaked at 2 hours (3036 ± 160.3 units/ml) and decreased thereafter. Conversely, levels of sPLA2 peaked at 48 hours (1.97 ± 0.64 ng/ml) and were increased further (14.02 ± 4.16 ng/ml) by pretreatment with anti-TNFα antibody. Cell accumulation was not affected by antibody pretreatment. These data indicate that the sPLA2 enzyme is involved in LPS-induced pleurisy. The enzyme seems not to be stimulated by TNFα which may be involved in the downregulation of sPLA2 in this model of inflammation

Human group II phospholipase A2 in normal and diseased intervertebral discs

AbstractWe measured calcium-dependent phospholipase A2 (PLA2) activity and immunoreactive group II PLA2 levels of 54 normal discs obtained from cadavers and 73 disc samples surgically obtained from patients with spinal disorders, including intervertebral disc herniations, spondylosis, and spondylolisthesis. Both cadaveric and surgical disc specimens contained about two-fold greater PLA2 activity than the ileal mucosa, one of the richest sources of group II PLA2. Discs of middle-aged cases had significantly higher activity than those of younger and elder cases. In cadaveric normal discs, calcium-dependent PLA2 activity was significantly higher in females than in males. Annulus fibrosus and nucleus pulposus contained the same PLA2 levels. In diseases discs, herniated fragments that had extruded or protruded out of the discs possessed lower activity than other parts of discs in the intervertebral space. Immunoreactive group II PLA2 levels of intervertebral discs closely correlated with PLA2 enzymatic activity. We purified a PLA2 from human intervertebral disc to homogeneity to further identify the isozymic nature of discal PLA2. Its NH2-terminal amino acid sequence and molecular weight were identical to those of human group II PLA2. Immunohistochemical analysis using a monoclonal anti-group II PLA2 antibody showed that in both annulus fibrosus and nucleus pulposus chondrocyte contained intense grou II PLA2 immunoreactivity in their cytoplasm, and that the matrix contained no substantial immunoreactivity. These results suggest that group II PLA2 in chondrocytes has important physiological roles in discal ordinary metabolism, maintaining discal homeostasis

Urate crystals induce macrophage PAF‑AH secretion which is differentially regulated by TGFβ1 and hydrocortisone

The aim of the present study was to establish the role of platelet‑activating factor acetyl hydrolase (PAF‑AH) in the resolution phase of gout using an established in vitro mononuclear cell model. The effects of signalling pathway inhibitors on PAF‑AH secretion, as well as the effects of the common treatments hydrocortisone and colchicine, an antibody against the anti‑inflammatory cytokine transforming growth factor β1 (TGFβ1), and the transcriptional inhibitor actinomycin D, were also investigated. The effect of recombinant PAF‑AH on cytokine secretion by these cells was also determined. Human peripheral blood‑derived monocytes were isolated and differentiated into macrophages. Monocytes and macrophages were stimulated with monosodium monohydrate urate (MSU) crystals or lipopolysaccharide in the presence or absence of AEG3482 [a c‑Jun N‑terminal kinase (JNK) inhibitor], MG132 (a proteasome inhibitor), hydrocortisone or colchicine. Cultures were then analysed for PAF‑AH secretion using ELISA. A 6‑fold upregulation of PAF‑AH secretion was observed following macrophage exposure to MSU crystals for 24 h (29.3±6 vs. 5.4±0.3 ng/ml unstimulated; P<0.05). Following 72 h, PAF‑AH levels decreased significantly (11.1±1.8; P<0.01). Secretion was further enhanced following pre‑treatment with the JNK protein kinase inhibitor AEG3482 prior to MSU crystal stimulation (P<0.05) and was abrogated when cells were preincubated with actinomycin D or the proteasome inhibitor MG132 (50, 100 and 200 µM). The addition of recombinant PAF‑AH (2.5‑10 ng/ml) to MSU crystal‑stimulated immature monocyte cultures significantly decreased pro‑inflammatory interleukin (IL)‑1β (unstimulated 687±124 vs. stimulated 113±30 pg/ml) and IL‑6 secretion (unstimulated 590±50 vs. stimulated 182±19 pg/ml). Treatment of MSU crystal‑stimulated macrophages with hydrocortisone (2 µM) also significantly decreased PAF‑AH release (P<0.05). Neutralising anti‑TGFβ1 addition decreased PAF‑AH dose‑dependently with the highest inhibition observed at 1 µg/ml (P<0.05). The results implicated that PAF‑AH may have an anti‑inflammatory role in the resolution phase of gout

Characterization of Serum Phospholipase A2 Activity in Three Diverse Species of West African Crocodiles

Secretory phospholipase A2, an enzyme that exhibits substantial immunological activity, was measured in the serum of three species of diverse West African crocodiles. Incubation of different volumes of crocodile serum with bacteria labeled with a fluorescent fatty acid in the sn-2 position of membrane lipids resulted in a volume-dependent liberation of fluorescent probe. Serum from the Nile crocodile (Crocodylus niloticus) exhibited slightly higher activity than that of the slender-snouted crocodile (Mecistops cataphractus) and the African dwarf crocodile (Osteolaemus tetraspis). Product formation was inhibited by BPB, a specific PLA2 inhibitor, confirming that the activity was a direct result of the presence of serum PLA2. Kinetic analysis showed that C. niloticus serum produced product more rapidly than M. cataphractus or O. tetraspis. Serum from all three species exhibited temperature-dependent PLA2 activities but with slightly different thermal profiles. All three crocodilian species showed high levels of activity against eight different species of bacteria

Control of mucosal polymicrobial populations by innate immunity

The gastrointestinal tract carries out the complex process of localizing the polymicrobial populations of the indigenous microbiota to the lumenal side of the GI mucosa while absorbing nutrients from the lumen and preventing damage to the mucosa. This process is accomplished through a combination of physical, innate and adaptive host defences and a ‘strategic alliance’ with members of the microbiota. To cope with the constant exposure to a diverse microbial community, the GI tract, through the actions of a number of specialized cells in the epithelium and lamina propria, has layers of humoral, physical and cellular defences that limit attachment, invasion and dissemination of the indigenous microbiota. However, the role of the microbiota in this dynamic balance is vital and serves as another level of ‘innate’ defence. We are just beginning to understand how bacterial metabolites aid in the control of potential pathogens within the microbiota and limit inflammatory responses to the microbiota, concepts that will impact our understanding of the biological effects of antibiotics, diet and probiotics on mucosal inflammatory responses.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72943/1/j.1462-5822.2009.01347.x.pd

Enzyme-responsive nanomaterials for controlled drug delivery

Enzymes underpin physiological function and exhibit dysregulation in many disease-associated microenvironments and aberrant cell processes. Exploiting altered enzyme activity and expression for diagnostics, drug targeting, and drug release is tremendously promising. When combined with booming research in nanobiotechnology, enzyme-responsive nanomaterials for controlled drug release have achieved significant development and been studied as an important class of drug delivery devices in nanomedicine. In this review, we describe enzymes such as proteases, phospholipase and oxidoreductases that serve as delivery triggers. Subsequently, we explore recently developed enzyme-responsive nanomaterials with versatile applications for extracellular and intracellular drug delivery. We conclude by discussing future opportunities and challenges in this area