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Lysophospholipid transacetylase in the regulation of PAF levels in human monocytes and macrophages

Luigi Servillo^*, Ciro Balestrieri^*, Alfonso Giovane^*, Paola Pari^*, Davide Palma^*, Giorgio Giannattasio, Massimo Triggiani and Maria Luisa Balestrieri^*,1

^* Department of Biochemistry and Biophysics, Second University of Naples; and

Division of Allergy and Clinical Immunology, University of Naples Federico II, Naples, Italy

¹Correspondence: Department of Biochemistry and Biophysics, Second University of Naples, via L. De Crecchio 7, Naples 80138, Italy. E-mail: mluisa.balestrieri{at}unina2.it

SPECIFIC AIM

The aim of this study is to investigate the role of the transacetylase activities (TA), namely lysophospholipid TA (TA_L), sphingosine (TA_S), and acetylhydrolase (AH), in the regulation of platelet-activating factor (PAF) levels during the maturation process of monocytes (Mo) into macrophages (Mf).

Principal findings

1. LPS and GM-CSF induce TA_L activity in human Mo
To determine the possible TA_L involvement in the regulation of the PAF levels in the mononuclear phagocyte system cells, we examined whether the TA activities were induced in Mo stimulated with LPS or granulocyte-macrophage colony-stimulating factor (GM-CSF), two agents known to activate inflammatory cells by stimulating the PAF metabolism. Results indicate that these two agonists stimulate not only the PAF biosynthesis catalyzed by the acetyl-coenzyme A acetyltransferase (AT), as already known, but, interestingly, also its degradation catalyzed by the TA_L. Specifically, the maximal TA_L activation occurs at concentration of 20 ng/ml and is approximately twofold (specific activity of the control was 0.96 nmol/min/mg protein). The time-course curve of the LPS-stimulated Mo shows an induction of the TA_L activity characterized by an early peak at 15 min and a delayed peak at 1 h, a pattern similar to the biphasic PAF accumulation. The highest induction of the TA_L activity (200% of the control) in Mo stimulated with GM-CSF for 15 min occurs at a concentration of 10 ng/ml (Fig. 1 ). The specific induction of the TA_L activity in activated Mo suggests that PAF is preferentially used for the synthesis of PAF analogues, rather than for the synthesis of N-acetyl-sphingosine (TA_S activity) or for its hydrolysis (AH activity). To characterize the molecular species of PAF-AH involved in the two peaks of enhanced TA_L activity detected during activation of Mo by LPS, we performed Western blot analysis of cell lysates with polyclonal antibodies against PAF-AH (II) and plasma PAF-AH, the two PAF-AH isoforms known to possess TA_L activity. Results indicate that both the early and late peak of LPS-induced TA activity are due to PAF-AH (II). Enzyme assays on cell lysates from Mo activated by GM-CSF for 15 min (10 ng/ml) or by LPS for 1 h (20 ng/ml) after immunoprecipitation with specific antibodies against PAF-AH (II) or plasma PAF-AH indicate that 76% of the TA_L activity was recovered in the fraction immunoprecipitated with PAF-AH (II) antibodies.

View larger version (15K): [in this window] [in a new window]   Figure 1. GM-CSF induces AT and TAL activities in human Mo. Adherent Mo were stimulated for 15 min with different concentration of GM-CSF in HBSS-10 mM HEPES. At the end of the incubations, AT, TAL, and AH activities were determined on the total cell homogenates as described in Materials and Methods. Results are means ± SE of 3 separate experiments in duplicate. P values were <0.01 for the AT and TAL activities in cells stimulated with GM-CSF (10 ng/ml) vs. control cells.

In contrast, when the cell lysates were immunoprecipitated with antibodies against plasma PAF-AH, the highest TA_L activity was still detected in the supernatant fraction (80% of the cell lysate activity). These data indicate that PAF-AH (II) is the molecular species of PAF-AH involved in both early and late peak of increased TA_L activity during activation of Mo by LPS and GM-CSF.

2. TA_L and AH activities increase during Mf differentiation
To determine the contribution of the TA activities in the mechanism responsible for the decreased PAF accumulation in Mf, we performed enzyme assays on the cell homogenates from Mf cultured for 10 days in presence of 20% FCS. Interestingly, we found that both TA_L and AH activities progressively increased during in vitro Mf differentiation. The increase of the TA_L activity during the differentiation process ranged from 180% (day 2) up to 334% of the control (day 7) and 410% of the control (day 10) and was more consistent than the increase of the AH activity (136, 252, and 320% of the control at days 2, 7, and 10, respectively; Fig. 2 ).

View larger version (13K): [in this window] [in a new window]   Figure 2. TAL and AH activities of the TA increase during Mf differentiation. Mf (2.5x106 cells/well) were cultured in complete RPMI 1640 supplemented with 20% FCS up to 10 days. At the end of the differentiation period, the cells were washed twice with HBSS-10 mM HEPES and the AT, TAL, and AH activities were determined on the total cell homogenates as described in Materials and Methods. Results are means ± SE of 5 separate experiments in duplicate. P values were <0.01 for the TAL and AH activities at day 10 vs. control cells.

The results from Western blot analysis with polyclonal antibodies against plasma PAF-AH and PAF-AH (II) showed that Mf accumulate plasma PAF-AH during differentiation, as indicated by the bands comigrating with recombinant plasma PAF-AH starting from day 5 of differentiation. These cells, concomitantly, express PAF-AH (II), which seems to be the predominant acetylhydrolase isoform. In addition, the Western blot pattern indicates additional bands recognized by the antibody anti-PAF-AH (II) slightly shifted at lower MW. Treatment of Mf lysates with alkaline phosphatase leads to changes of the immunoreactive pattern, which showed only bands comigrating with standard PAF-AH (II). Together these results suggest that the increase of TA_L and AH activities in Mf during differentiation can be ascribed to both PAF-AH (II), which resulted to be activated by phosphorylation, and to accumulation of plasma PAF-AH, an acetylhydrolase known to be regulated at transcriptional level.

3. ERKs and p38 MAP kinase pathways mediate TA_L induction in Mo but not in Mf
To investigate the role of ERKs and p38 MAP kinase in the TA_L activation in Mo/Mf, we employed selective inhibitors against p38 MAP kinase and ERKs, i.e., SB 203580 and PD 98059, respectively. The results indicate that TA_L specific activity induced by LPS or GM-CSF returned near to control values when Mo were pretreated with p38 MAP kinase or ERKs inhibitors. Moreover, when the PAF biosynthesis was determined in intact Mo preincubated with PD98059 or SB 203580, both compounds reduced the LPS-stimulated 1-radyl-[³H]acetyl-GPC production. Similarly, these two inhibitors reduced the 1-radyl-2-[³H]acetyl-GPC production induced by stimulation with GM-CSF to near control. On the whole these results indicate that in Mo both PAF biosynthesis (AT activity) and degradation (TA_L activity) are regulated by p38/ERKs pathways.

Instead, in Mf, both MAP kinase inhibitors completely inhibited the activation of the AT, whereas no significant effect was observed on the TA_L and AH activities. These results were also confirmed by experiments performed with recombinant, constitutively activated p38, ERK-1, or ERK-2 kinases in a cell-free system.

4. Translocation of the PAF-AH(II) from cytosol to membranes
PAF-AH (II) is known to be localized in both membrane and cytosol fractions and to translocate to the membrane in response to changes in the cell redox state and to direct/indirect phosphorylation. To investigate whether agonist stimulation caused translocation of the PAF-AH (II), we analyzed the subcellular distribution of PAF-AH (II) in membrane fraction and in cytosol fraction upon treatment of Mo with LPS and GM-CSF. Results from Western blot analysis of the subcellular fractions indicate that PAF-AH (II), evenly distributed in the cytosol and membrane fractions of control Mo, translocated to the membranes upon LPS and M-CSF stimulation. When the TA_L and AH activities were measured in the membrane fraction, translocation of the PAF-AH (II) was specifically reflected in an increase of the membrane-associated TA_L activity but not AH activity. These data indicated that stimulation of Mo induces translocation of PAF-AH (II) to the cell membrane and that this process can, therefore, be considered an event involved in the differential regulation of the TA_L and AH activities of PAF-AH (II).

CONCLUSIONS

PAF is one of the bioactive molecules produced by Mo and Mf. Although stimulated Mo release a large fraction of newly synthesized PAF, Mf are a major source of plasma PAF-AH. The balance of the PAF production and degradation during the differentiation of Mo into Mf is of pathophysiological importance; thus it is carefully controlled during this process. It has been suggested that the PAF-AH released by Mf may influence local inflammatory events by degrading oxidized phospholipids and PAF and that PAF-AH may be transported to the blood, where it serves to limit the PAF half-life in plasma. The mechanisms regulating PAF concentration, essential in the control of pathological inflammation, are still matter of studies since alterations of the enzymes that control PAF levels result in a number of pathologies.

This study offers the first demonstration that TA_L is involved in the regulation of PAF levels in Mo and Mf. Moreover, since the definition of the signal transduction pathway(s) responsible for the regulation of PAF levels significantly contribute to effective therapeutic strategies to control inflammatory diseases, this study also describes the key events in the TA_L regulation, i.e., p38/ERKs-mediated phosphorylation, subcellular translocation, and the changes that occur when Mo were in vitro differentiated into Mf.

Specifically, we report that in Mo LPS or GM-CSF increased the TA_L activity. Western blot analysis and enzyme assays on immunoprecipitates revealed that the increased activity was to be ascribed to PAF-AH (II) and that both translocation from cytosol to membranes and p38/ERKs-mediated phosphorylation regulate the enzyme activation. In Mf differentiated in vitro from Mo by incubation with FCS, an increase of both TA_L and AH activities was observed. Moreover, activation of ERKs and p38 MAP kinase was not required for the up-regulation of PAF-AH (II) in differentiated Mf. The differences observed in Mf as compared to Mo can be explained by 1) p38/ERKs-independent phosphorylation of PAF-AH (II) and 2) appearance of plasma PAF-AH in the course of Mf differentiation (Fig. 3 ). Additional studies are required for elucidation of the kinase(s) responsible for the phosphorylation of PAF-AH (II) in Mf. In conclusion, the identification of lysophospholipid transacetylase in human Mo and in vitro derived Mf highlights a new way for the modulation of PAF levels in inflammatory cells.

View larger version (23K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5059fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5059fjev1 20/7/1015    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Servillo, L. Articles by Balestrieri, M. L. Search for Related Content PubMed PubMed Citation Articles by Servillo, L. Articles by Balestrieri, M. L.