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Pseudomonas aeruginosa has two well-characterized quorum-sensing systems, Las and Rhl. These systems are composed of LuxR-type proteins, LasR and RhlR, and two acyl homoserine lactone (AHL) synthases, LasI and RhlI. LasI catalyzes the synthesis of N-(3-oxododecanoyl)homoserine lactone (3O-C 12 -HSL), whereas RhlI catalyzes the synthesis of N-butyryl-homoserine lactone. There is little known about the importance of AHLs in vivo and what effects these molecules have on eukaryotic cells. In order to understand the role of AHLs in vivo, we first tested the effects that deletions of the synthase genes in P. aeruginosa had on colonization of the lung. We demonstrate that in an adult mouse acute-pneumonia model, deletion of the lasI gene or both the lasI and rhlI genes greatly diminished the ability of P. aeruginosa to colonize the lung. To determine whether AHLs have a direct effect on the host, we examined the effects of 3O-C 12 -HSL injected into the skin of mice. In this model, 3O-C 12 -HSL stimulated a significant induction of mRNAs for the cytokines interleukin-1␣ (IL-1␣) and IL-6 and the chemokines macrophage inflammatory protein 2 (MIP-2), monocyte chemotactic protein 1, MIP-1␤, inducible protein 10, and T-cell activation gene 3. Additionally, dermal injections of 3O-C 12 -HSL also induced cyclooxygenase 2 (Cox-2) expression. The Cox-2 enzyme is important for the conversion of arachidonic acid to prostaglandins and is associated with edema, inflammatory infiltrate, fever, and pain. We also demonstrate that 3O-C 12 -HSL activates T cells to produce the inflammatory cytokine gamma interferon and therefore potentially promotes a Th1 environment. Induction of these inflammatory mediators in vivo is potentially responsible for the significant influx of white blood cells and subsequent tissue destruction associated with 3O-C 12 -HSL dermal injections. Therefore, the quorum-sensing systems of P. aeruginosa contribute to its pathogenesis both by regulating expression of virulence factors (exoenzymes and toxins) and by inducing inflammation.

Peroxisome proliferator‐activated receptor (PPAR)‐γ is a nuclear hormone receptor that serves as a trans factor to regulate lipid metabolism. Intense interest is focused on PPAR‐γ and its ligands owing to its putative role in adipocyte differentiation. Little is known, however, about the functions of PPAR‐γ in the immune system, especially in T lymphocytes. We demonstrate that both naive and activated ovalbumin‐specific T cells from DO11.10‐transgenic mice express PPAR‐γ mRNA and protein. In order to determine the function of PPAR‐γ, T cells were stimulated withphorbol 12‐myristate 13‐acetate and ionomycin or antigen and antigen‐presenting cells. Simultaneous exposure to PPAR‐γ ligands (e. g. 15‐deoxy‐Δ12, 14‐prostaglandin J2, troglitazone) showed drastic inhibition of proliferation and significant decreases in cell viability. The decrease in cell viability was due to apoptosis of the T lymphocytes, and occurred only when cells were treated with PPAR‐γ, and not PPAR‐α agonists, revealing specificity of this response for PPAR‐γ. These observations suggest that PPAR‐γ agonists play an important role in regulating T cell‐mediated immune responses by inducing apoptosis. T cell death via PPAR‐γ ligation may act as a potent anti‐inflammatory signal in the immune system, and ligands could possibly be used to control disorders in which excessive inflammation occurs.

Mast cells, platelets, and some macrophages are abundant sources of PGD2 and its active metabolite 15-deoxy-Δ12,14-PGJ2 (15-d-PGJ2). The lipid mediator 15-d-PGJ2 regulates numerous processes, including adipogenesis, apoptosis, and inflammation. The 15-d-PGJ2 has been shown to both inhibit as well as induce the production of inflammatory mediators such as TNF-α, IL-1β, and cyclooxygenase, mostly occurring via a nuclear receptor called peroxisome proliferator-activated receptor-γ (PPAR-γ). Data concerning the effects of 15-d-PGJ2 on human T cells and immune regulation are sparse. IL-8, a cytokine with both chemotactic and angiogenic effects, is produced by T lymphocytes following activation. Whether 15-d-PGJ2 can regulate the production of IL-8 in T cells in unknown. Interestingly, 15-d-PGJ2 treatment of unstimulated T cells induces cell death. In contrast, in activated human T lymphocytes, 15-d-PGJ2 does not kill them, but induces the synthesis of IL-8. In this study, we report that 15-d-PGJ2 induced a significant increase in both IL-8 mRNA and protein from activated human T lymphocytes. The induction of IL-8 by 15-d-PGJ2 did not occur through the nuclear receptor PPAR-γ, as synthetic PPAR-γ agonists did not mimic the IL-8-inducing effects of 15-d-PGJ2. The mechanism of IL-8 induction was through a mitogen-activated protein kinase and NF-κB pathway, as inhibitors of both systems abrogated IL-8 protein induction. Therefore, 15-d-PGJ2 can act as a potent proinflammatory mediator in activated T cells by inducing the production of IL-8. These findings show the complexity with which 15-d-PGJ2 regulates T cells by possessing both pro- and anti-inflammatory properties depending on the activation state of the cell. The implications of this research also include that caution is warranted in assigning a solely anti-inflammatory role for 15-d-PGJ2.

SUMMARYProstaglandin D 2 (PGD 2 ) is abundantly produced by mast cells, platelets, and alveolar macrophages and has been proposed as a key immunoregulatory lipid mediator. 15-Deoxy-D 12,14 -PGJ 2 (15-d-PGJ 2 ), a key PGD 2 metabolite, is under intense study as a potential anti-in¯ammatory mediator. Little is known about PGD 2 or the role of 15-d-PGJ 2 , if any, in regulating the activities of human T lineage cells. In this report we demonstrate that both PGD 2 and 15-d-PGJ 2 have potent antiproliferative effects, and in fact kill human T lymphocyte lines derived from malignant cells by an apoptotic mechanism. Interestingly, normal human T cells were not similarly affected. Although the T lymphocyte lines express mRNA for the PGD 2 receptor (DP-R), a potent DP receptor agonist, BW245C, did not inhibit the proliferation or viability of the cells, suggesting an alternative mechanism of action. PGD 2 and 15-d-PGJ 2 can bind to the peroxisome proliferator activated receptor-c (PPAR-c) which is implicated in lipid metabolism and apoptosis. Exposure to synthetic PPAR-c ligands (e.g. ciglitazone, troglitazone) mimicked the inhibitory responses of PGD 2 and 15-d-PGJ 2 , and induced apoptosis in the transformed T cells consistent with a PPAR-cdependent mechanism. These observations suggest that PPAR-c ligands (which may include PGD 2 ) provide strong apoptotic signals to transformed, but not normal T lymphocytes. Thus, the ef®cacy of utilizing PPAR-c and its ligands as therapeutics for human T cell cancers needs to be further evaluated.

Prostaglandins of the E‐series stimulate B lymphocytes by enhancing immunoglobulin‐class switching and antibody production. Little is known about whether or not other prostaglandins affect B lineage cells and perhaps counterbalance the stimulatory effects of PGE2. PGD2 is a major product of cyclooxygenase in bone marrow and in macrophages, suggesting a role for this lipid product in immunological responses. PGD2 undergoes dehydration to the biologically active prostaglandin 15‐deoxy‐Δ12,14‐PGJ2 (15d‐PGJ2) that binds to the nuclear receptor known as peroxisome proliferator‐activated receptor gamma (PPAR‐γ). We found that normal mouse B cells and a Wvariety of B lymphoma cells (e.g., 70Z/3, WEHI‐231, CH12, and J558) express PPAR‐γmRNA and the 67‐kDa PPAR‐γ protein. 15d‐PGJ2 had a dose‐dependent antiproliferative/cytotoxic effect on normal and malignant B cells, as shown by 3H‐thymidine and MTT assays. Only PPAR‐γ agonists (i.e., thiazolidinediones) mimicked the effect of 15d‐PGJ2 on B lineage cells, indicating that the mechanism by which 15d‐PGJ2 negatively affects B lineage cells involves PPAR‐γ. The mechanism whereby PPAR‐γ agonists induced cytotoxicity is via apoptosis, as shown by Annexin V assays. PPAR‐γ agonists may serve as a counterbalance to the stimulating effects of PGE2, which promotes B‐cell differentiation. The use of prostaglandins, such as 15d‐PGJ2, and synthetic PPAR‐γ agonists to induce apoptosis in B lineage cells may lead to the development of therapies for fatal PGE2‐resistant B lymphomas.

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