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PLIF induces IL-10 production in monocytes: a calmodulin-p38 mitogen-activated protein kinase-dependent pathway¹

MUAYAD A. ZAHALKA^*, VIVIAN BARAK, LEONID TRAUB^* and CHAYA MOROZ^*,2

^* Laboratory of Molecular Immunology, Felsenstein Medical Research Center, Sackler School of Medicine, Tel Aviv University, Rabin Medical Center, Petach-Tikva, Israel; and
Immunology-Oncology Laboratory, Hadassah Medical Center, Jerusalem, Israel

²Correspondence: Laboratory of Molecular Immunology, Felsenstein Medical Research Center, Sackler School of Medicine, Tel Aviv University, Rabin Medical Center, Petach-Tikva 49100, Israel. E-mail: hmoroz{at}post.tau.ac.il

SPECIFIC AIMS

We recently reported cloning of the human pregnancy-related factor PLIF (placenta immunomodulatory ferritin). PLIF is composed of a ferritin heavy chain-like domain and a novel bioactive domain named C48. PLIF as well as C48 inhibited T cell proliferation after immune activation. In this study, we aimed to characterize the physiologic function of PLIF resulting in the immunosuppressive effect and further identify the molecular basis leading to its bioactivity.

PRINCIPAL FINDINGS

1. C48 induces immune activation-independent IL-10 production
The effect of recombinant PLIF and C48—the bioactive domain of PLIF—on cytokine network was studied after T cell activation in mixed lymphocytes culture (MLC). Changes in key cytokines after treatment with C48 were monitored in MLC and in nonactivated peripheral blood mononuclear cell (PBMC) cultures. Among the cytokines tested, IL-10 was found to be markedly elevated in the C48-treated MLC (Fig. 1 B). However, high levels of IL-10 were also measured in nonactivated PBMC treated with C48 (Fig. 1A ), indicating that C48 induced IL-10 production in PBMCs independent of immune activation. Other cytokines tested did not significantly change after C48 treatment. Similar results were obtained when C48 was substituted with full-length PLIF. In control experiments, treatment of PBMCs with the glutathione-S-transferase (GST) part of C48-GST fusion protein did not result in elevation of IL-10 or any other cytokines.

View larger version (15K): [in this window] [in a new window]   Figure 1. Effect of C48 treatment on different cytokine production in PBMCs. PBMC cultures were treated with C48 or untreated and subsequently stimulated with allogeneic cells (B) or unstimulated (A). 24 h later, supernatants were collected and tested for the indicated cytokines. Fold cytokine increase was calculated as the amount of cytokine in C48-treated culture divided into the corresponding untreated.

2. C48 targets monocytes in induction of IL-10 production
To identify the C48-induced IL-10 producing cells, we isolated monocyte and T cell fractions from PBMCs by negative selection using an antibody-based technique.Both fractions as well as PBMCs were cultured in the presence or absence of C48 for 24 h. The results indicated that monocytes but not T cells were the IL-10 producers after C48 stimulation.

3. C48 activates ERK1,2 and p38 MAP kinases in monocytes
Next we studied the signaling events initiated by C48 in monocytes that lead to induction of IL-10 production. We found that C48 induced sustained activation of both ERK1,2 (extracellular-regulated kinases 1, 2) and p38 MAP (mitogen-activated protein) kinases. In comparison, no marked activation of these MAP kinases was observed in T cells in response to C48 treatment.

4. p38 MAP kinase mediates C48-induced IL-10 production
We investigated whether ERK1,2 and p38 MAP participate in the signaling cascade leading to IL-10 production. This was performed by testing the effect of appropriate inhibitors for each of the indicated MAP kinases on C48-induced IL-10 production. We found that SB203580, a p38 MAP kinase inhibitor, completely inhibited C48-induced IL-10 production in monocytes. On the other hand, inhibition of ERK1,2 activation by the MAP kinase kinase inhibitor PD98059 did not inhibit C48-stimulated IL-10 production but rather enhanced it. The results suggest that p38 but not ERK MAP kinase-dependent pathway mediates C48-induced IL-10 production. Moreover, the increased IL-10 production in the presence of PD98059 may reflect a down-regulatory effect of the ERK MAP kinase on the p38 signaling pathway induced by C48.

5. C48-induced IL-10 production is calcium/calmodulin dependent
To further characterize the C48-initiated signaling pathway that leads to induction of IL-10 production, we tested the ability of some signal transduction inhibitors to block C48-induced IL-10 production and MAP kinases activation in monocytes. It was found that W-7, the calmodulin antagonist, completely abolished IL-10 production stimulated by C48. On the other hand, the calcium-chelating agent EGTA had partial inhibitory effect on this cytokine production (Fig. 2 A). Other inhibitors like the protein kinase A inhibitor, H-89, and the protein kinase C inhibitor bisindolylmaleimide I had a very limited inhibitory effect on this process (Fig. 2A ).

View larger version (23K): [in this window] [in a new window]   Figure 2. Effect of several signal transduction inhibitors on C48-induced IL-10 production and MAP kinases activation. Monocyte cultures were pretreated for 2 h with each inhibitor or unpretreated, then stimulated with C48. A) Cultures were maintained for 24 h after stimulation with C48 and supernatants were assayed for IL-10 levels. Results are presented as % compared with corresponding cultures without inhibitors. B) Pretreated cultures were stimulated with C48 for 30 min and lysed. Lysates were immunoblotted and tested with appropriate mAbs to activated forms of ERK1,2 and p38 MAP kinases. The inhibitors used are EGTA (1 mM), W-7 (50 µM), bisindolylmaleimide I (100 nM), H-89 (100 nM).

Further, we demonstrated that C48-induced activation of both MAP kinases is blocked by pretreatment with W-7 (Fig. 2B ), suggesting that both kinases are downstream to a calmodulin-dependent pathway similar to IL-10 production.

CONCLUSION AND SIGNIFICANCE

Our results suggest that the inhibitory effect of PLIF on T cell proliferation may result at least in part from its interaction with target molecule(s) on antigen-presenting cells. Such interaction leads to activation of a calmodulin-p38 MAP kinase-dependent signaling pathway that mediates induction of IL-10 production. IL-10 will in turn provide an immunosuppressive effect on T cell activation via inhibition of CD28 tyrosine phosphorylation. It is therefore suggested that the physiologic bioactivity of PLIF present at high levels in the fetal-maternal interface and maternal blood throughout gestation is in part to induce IL-10 production. Moreover, IL-10 has been postulated to play a role in the embryo's protection from maternal immune attack.

Our current findings of the mechanism of action of the novel immune modulator PLIF may contribute to understanding the enigma regarding the embryonic protection against maternal immune rejection.

On a practical level, the unique characteristic of PLIF marks its great therapeutic potential in clinical situations like autoimmune diseases and transplantation, where anti-inflammatory and immunosuppressive effects are beneficial.

View larger version (15K): [in this window] [in a new window]   Figure 3. Schematic diagram of the hypothesized bioactivity and mechanism of action of C48 (PLIF).

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0960fje; to cite this article, use FASEB J. (March 28, 2003) 10.1096/fj.02-0960fje

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