Evidence that 85 kDa phospholipase A2 is not linked to CoA-independent transacylase-mediated production of platelet-activating factor in human monocytes.

Platelet-activating factor (PAF) production is carefully controlled in inflammatory cells. The specific removal of arachidonate (AA) from 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (GPC), thought to be mediated by CoA-independent transacylase (CoA-IT), is required to generate the PAF precursor 1-O-alkyl-2-lyso-GPC in human neutrophils. Exposure of A23187-stimulated human monocytes to the CoA-IT inhibitors SK&F 98625 and SK&F 45905 inhibited PAF formation (IC50s of 10 and 12 microM, respectively), indicating that these cells also need CoA-IT activity for PAF production. Because CoA-IT activity transfers arachidonate to a 2-lyso phospholipid substrate, its activity is obligated to an sn-2 acyl hydrolase to form the 2-lyso phospholipid substrate. SB 203347, an inhibitor of 14 kDa phospholipase A2 (PLA2), and AACOCF3, an inhibitor of 85 kDa PLA2, both inhibited AA release from A23187-stimulated human monocytes. However, AACOCF3 had no effect on A23187-induced PAF formation at concentrations as high as 3 microM. Further, depletion of 85 kDa PLA2 using antisense (SB 7111, 1 microM) had no effect on PAF production, indicating a lack of a role of 85 kDa PLA2 in PAF biosynthesis. Both SB 203347 and the 14 kDa PLA2 inhibitor scalaradial blocked PAF synthesis in monocytes (IC50s of 2 and 0.5 microM, respectively), suggesting a key role of 14 kDa PLA2 in this process. Further, A23187-stimulated monocytes produced two forms of PAF: 80% 1-O-alkyl-2-acetyl-GPC and 20% 1-acyl-2-acetyl-GPC, which were both equally inhibited by SB 203347. In contrast, inhibition of CoA-IT using SK&F 45905 (20 microM) had a greater effect on the production of 1-O-alkyl (-80%) than of 1-acyl (-14%) acetylated material. Finally, treatment of U937 cell membranes with exogenous human recombinant (rh) type II 14 kDa PLA2, but not rh 85 kDa PLA2, induced PAF production. Elimination of membrane CoA-IT activity by heat treatment impaired the ability of 14 kDa PLA2 to induce PAF formation. Taken together, these results suggest that a 14 kDa PLA2-like activity, and not 85 kDa PLA2, is coupled to monocyte CoA-IT-induced PAF production.

The human polo-like kinase, PLK, regulates cdc2/cyclin B through phosphorylation and activation of the cdc25C phosphatase.

Entry into mitosis by mammalian cells is triggered by the activation of the cdc2/cyclin B holoenzyme. This is accomplished by the specific dephosphorylation of key residues by the cdc25C phosphatase. The polo-like kinases are a family of serine/threonine kinases which are also implicated in the control of mitotic events, but their exact regulatory mechanism is not known. Recently, a Xenopus homologue, PLX1, was reported to phosphorylate and activate cdc25, leading to activation of cdc2/cyclin B. Jurkat T leukemia cells were chemically arrested and used to verify that PLK protein expression and its phosphorylation state is regulated with respect to cell cycle phase (i.e., protein is undetectable at G1/S, accumulates at S phase and is modified at G2/M). Herein, we show for the first time that endogenous human PLK protein immunoprecipitated from the G2/M-arrested Jurkat cells directly phosphorylates human cdc25C. In addition, we demonstrate that recombinant human (rh) PLK also phosphorylates rhcdc25C in a time- and concentration-dependent manner. Phosphorylation of endogenous cdc25C and recombinant cdc25C by PLK resulted in the activation of the phosphatase as assessed by dephosphorylation of cdc2/cyclin B. These data are the first to demonstrate that human PLK is capable of phosphorylating and positively regulating human cdc25C activity, allowing cdc25C to dephosphorylate inactive cdc2/cyclin B. As this event is required for cell cycle progression, we define at least one key regulatory mode of action for human PLK in the initiation of mitosis.

Anti-human RP105 sera induces lymphocyte proliferation.

Cellular environment dictates whether antigen binding to the B lymphocyte receptor together with co-stimulatory molecules will result in proliferation, anergy, or apoptosis. Murine RP105 is a member of the leucine-rich repeat family of proteins, which is specifically expressed on mature B cells. Monoclonal antibodies to the murine RP105 induce proliferation and protect B cells from apoptosis, suggesting an important regulatory role in murine B lymphocyte function. We identified a human RP105 homolog and mapped the gene to chromosome 5q12.3-13.1. Tissue distribution analysis shows that the transcript is found predominately in lymphoid tissues including spleen, tonsils, appendix, and peripheral blood leukocytes. Polymerase chain reaction analysis of isolated primary human cell populations confirms that mRNA exists in spleen B lymphocytes and monocytes but not T lymphocytes. Western blot analysis demonstrates specific expression of human RP105 in human B lymphocytes. Murine anti-human RP105 sera was generated using DNA immunization. The antisera contained antibodies that recognized and bound to human B lymphocytes from both spleen and peripheral blood as assessed by flow cytometry. Assessment of biological function showed that human peripheral blood leukocytes incubated with anti-RP105 sera were induced to proliferate as measured by tritiated thymidine incorporation. Moreover, anti-CD40 and interleukin-4-treated cells but not those exposed to anti-RP105 sera produced soluble CD23, suggesting distinct functional roles. This is the first demonstration of both the existence of RP105 protein on human B lymphocytes and its role in the regulation of B lymphocyte activation.

Human calcium-independent phospholipase A2 mediates lymphocyte proliferation.

Activation of lymphocytes induces blastogenesis and cell division which is accompanied by membrane lipid metabolism such as increased fatty acid turnover. To date little is known about the enzymatic mechanism(s) regulating this process. Release of fatty acids such as arachidonic acid requires sn-2-deacylation catalyzed by a class of enzymes known as phospholipases A(2) (PLA(2), EC ). Herein, we confirm that human peripheral blood B or T lymphocytes (PBL) do not possess measurable levels of 85-kDa PLA(2) as assessed by Western immunoblot. Low levels of 14-kDa PLA(2) protein and activity were detectable in the particulate fraction of PBL and Jurkat cells. Western immunoblot analysis indicates that PBLs possess the calcium-independent PLA(2) (iPLA(2)) protein. Calcium-independent sn-2-acylhydrolytic activity was measurable in PBL cytosols and could be inhibited by the selective iPLA(2) inhibitor bromoenol lactone. Mitogen activation of PBLs resulted in maintenance of activity levels which remained constant over 72 h suggesting an important role for iPLA(2) in this proliferative process. Indeed, evaluation of iPLA(2) activity in cell cycle-arrested Jurkat T cell fractions revealed the highest iPLA(2) levels occurring at the G(2)/M phase. Addition of the iPLA(2) inhibitors, bromoenol lactone, or arachidonyl trifluoromethyl ketone (AAOCF(3)), inhibited both mitogen-induced PBL as well as Jurkat T cell proliferation. Moreover, specific depletion of iPLA(2) protein by antisense treatment also resulted in marked suppression of cell division. Inhibition of Jurkat cell proliferation was not associated with arrest at a particular phase of the cell cycle nor was it associated with apoptosis as assessed by flow cytometry. These findings provide the first evidence that iPLA(2) plays a key role in the lymphocyte proliferative response.

Manipulation of distinct NFkappaB proteins alters interleukin-1beta-induced human rheumatoid synovial fibroblast prostaglandin E2 formation.

Interleukin 1beta (IL-1beta) up-regulates human rheumatoid synovial fibroblast (RSF) 85-kDa phospholipase A2 (PLA2) and mitogen-inducible cyclooxygenase (COX) II. Promoter regions for these genes contain a motif that closely resembles the "classic" NFkappaB consensus site. Immunoblot analysis identified NFkappaB1 (p50), RelA (p65), and c-Rel in RSF. Upon IL-1beta-stimulation, p65 and c-Rel but not p50 protein levels were reduced suggesting nuclear translocation. IL-1beta-induced RSF nuclear extracts contained a p65-containing complex, which bound to the classical NFkappaB consensus motif. An NFkappaB classical oligonucleotide decoy produced a concentration-dependent decrease in IL-1-stimulated PGE2 production (IC50 = approximately 2 microM), indicating a role of NFkappaB. Utilization of antisense technology showed that p65 but not p50 or c-Rel mediated IL-1beta-stimulated PGE2 formation. Treated RSF could not transcribe COX II or 85-kDa PLA2 mRNA, which reduced their respective proteins. Interestingly, stimulated IL-8 production was not inhibited by the classical NFkappaB decoy but was reduced by treatment with antisense to both p65 and c-Rel supporting preferential binding of c-Rel-p65 to the "alternative" IL-8 kappaB motif. Taken together, these data provide the first direct evidence for a role of p65 in COX II and 85-kDa PLA2 gene induction and support the IL-1 activation and participation of distinct NFkappaB protein dimers in RSF prostanoid and IL-8 formation.

Modulation of human monocyte activities by tranilast, SB 252218, a compound demonstrating efficacy in restenosis.

Tranilast (SB 252218) is a compound initially identified as an anti-atopic agent. Recently the compound has demonstrated clear beneficial effects in animal models of restenosis. Here we confirm tranilast has broad and profound effects on human monocytes, which could contribute to the vascular antifibrotic activity. Tranilast exhibited significant immunomodulatory activity inhibiting endotoxin-induced prostaglandin E(2) (PGE(2); IC(50) = approximately 1-20 microM), thromboxane B(2) (IC(50) = approximately 10-50 microM), transforming growth factor-beta1 (TGF-beta1; IC(50) = approximately 100-200 microM), and interleukin-8 (IC(50) = approximately 100 microM) formation, but had no effect on tumor necrosis factor-alpha. Interleukin-12 and -18-induced interferon-gamma formation by monocytes was also attenuated by tranilast. A23187-induced monocyte leukotriene C(4) or PGE(2) formation was inhibited by tranilast at IC(50) values of 10-40 microM and 2-20 microM, respectively, incubated with or without exogenous arachidonic acid. Interestingly, tranilast (up to 1000 microM) had no direct effects on cyclooxygenase I or II activity, nor did it have significant effects on human type IIA 14 kDa or type IV 85 kDa phospholipase A(2) activity. Furthermore, tranilast had no effect on endotoxin-induced cyclooxygenase II protein expression, suggesting tranilast modulates eicosanoid production and release by an as yet unidentified mechanism. Alternatively, the expression of TGF-beta1 was inhibited by tranilast but found to be due in part to inhibition of PGE(2) because exogenous PGE(2) could abrogate tranilast-mediated inhibition of TGF-beta1. Taken together, although a reported direct inhibitor of fibroblast proliferation, we show tranilast also attenuates the proinflammatory activity of human monocytes, adding to its potential efficacy as a therapeutic agent in restenosis.

Differential effects of SB 242235, a selective p38 mitogen-activated protein kinase inhibitor, on IL-1 treated bovine and human cartilage/chondrocyte cultures.

The p38 MAP kinase inhibitor, SB 242235, was evaluated for its effects on the metabolism of bovine and human cartilage and primary chondrocyte cultures. SB 242235 had no effect on proteoglycan synthesis (PG) in bovine articular cartilage explants (BAC), as measured by [(35)S]-sulfate incorporation into glycosaminoglycans (GAGs). In addition, the compound had no effect on IL-1 alpha-induced GAG release from these cultures. However, there was a potent, dose-dependent inhibition of nitric oxide (NO) release from IL-1 alpha-stimulated BAC with an IC(50)of approximately 0.6 microM, with similar effects observed in primary chondrocytes. The effect on BAC was time dependent, and mechanistically did not appear to be the result of inhibition of protein kinase C (PKC), protein kinase A (PKA) or MEK-1. The effect on NO release in bovine chondrocytes was at the level of inducible nitric oxide synthase (iNOS) gene expression, which was inhibited at similar concentrations as nitrite production. In primary human chondrocytes, IL-1 beta induction of p38 MAP kinase was inhibited by SB 242235 with an IC(50)of approximately 1 microM. Surprisingly, however, treatment of IL-beta-stimulated human cartilage or chondrocytes with SB 242235 did not inhibit either NO production or the induction of iNOS. On the other hand, the natural product hymenialdisine (HYM), a protein tyrosine kinase (PTK) inhibitor, inhibited NO production and iNOS in both species. In contrast to the differential control of iNOS, PGE(2)was inhibited by SB 242235 in both IL-1-stimulated bovine and human chondrocyte cultures. These studies indicate that there are species differences in the control of iNOS by p38 inhibitors and also that different pathways may control IL-1-induced proteoglycan breakdown and NO production.