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

Abstract

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‐α, IL‐6 and IL‐12 was determined by ELISA. Group IA, IB and IIA sPLA2 induced a concentration‐dependent release of TNF‐α and IL‐6 from bloodmonocytes. These sPLA2 activated IL‐12 production only in monocytes preincubated with IFN‐γ. Group IA and IIA sPLA2 also induced TNF‐α and IL‐6 release from synovial fluid monocytes. TNF‐α 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 theproinflammatory activity of sPLA2 released during inflammatory diseases.

Activation of Cytokine Production by Secreted Phospholipase A2 in Human Lung Macrophages Expressing the M-Type Receptor

“…In three experiments, at a concentration of 10 g/ml, hGX and bvGIII, but not hGIB, induced the release of a significant amount of AA from HLM (percentage of total cellular [ 3 H]AA released in 1 h: hGIB, 3.1 Ϯ 0.7%; bvGIII, 6.0 Ϯ 1.1% ( p Ͻ 0.05 vs unstimulated); hGX, 6.6 Ϯ 1.1 ( p Ͻ 0.05 vs unstimulated); unstimulated, 2.9 Ϯ 0.5). These results are in line with earlier studies showing that hGX and bvGIII, which bind to the extracellular face of mammalian cell membranes, but not hGIB, efficiently release [ 3 H]AA when added exogenously to intact mammalian cells (36,41,44). Our data demonstrate that the capacity of each sPLA 2 to mobilize AA in macrophages is unrelated to their ability to induce cytokine production.…”

“…Macrophages were incubated (6 h, 37°C) with increasing concentrations (0.1-10 g/ml) of hGIB or hGX and with bvGIII, an isoform that does not induce TNF-␣ and IL-6 production from human blood monocytes (41). Both hGIB and hGX induced a concentration-dependent release of TNF-␣ and IL-6 (Fig.…”

“…At the end of the incubation, RNA was isolated by the TRIzol technique (Invitrogen Life Technologies). RT-PCR was performed with target-specific primers for TNF-␣ and IL-6 as previously described (41). RNA was normalized for the constitutive marker gene ␤-actin.…”

“…Adherent macrophages (4 ϫ 10 6 /2 ml) were incubated (37°C) in RPMI 1640 alone or with hGX (10 g/ml) for different time periods (3-12 h). At the end of the incubation, RNA was isolated using the SV96 total RNA isolation system (Promega) according to the manufacturer's instructions and was treated with RNase-free DNase I. RT was performed as previously described (41). Real-time quantitative PCR was performed on the iCycler (Bio-Rad) using the PE-SYBR Green PCR kit (Applied Biosystems).…”

In Vivo Protective Role of Human Group IIA Phospholipase A2 against Experimental Anthrax

“…Clearly this does not exclude the potential implication of an indirect effect via modifications of the immune system per se, particularly through the interaction with a receptor on certain cell types (40). Several studies with various sPLA 2 s, including human group IIA, have shown that these sPLA 2 s can trigger iNOS induction (41), COX-2 induction (42) and IL-6, IL-8, and TNF-␣ cytokine secretion (43), through a mechanism involving ERK1/2 (41,44). However, in our study, sPLA 2 -IIA was found effective even against infection with a B. anthracis strain secreting the toxins that are known to block the MAPK cascade (ERK/P38) through MEK cleavage (45,46); this observation suggests a predominant direct bactericidal effect.…”