Richard P. Phipps

Prostaglandins as modulators of immunity

Prostaglandins are potent lipid molecules that affect key aspects of immunity. The original view of prostaglandins was that they were simply immunoinhibitory. This review focuses on recent findings concerning prostaglandin E2 (PGE2) and the PGD2 metabolite 15-deoxy-Delta(12,14)-PGJ2, and their divergent roles in immune regulation. We will highlight how these two seminal prostaglandins regulate immunity and inflammation, and play an emerging role in cancer progression. Understanding the diverse activities of these prostaglandins is crucial for the development of new therapies aimed at immune modulation.

A new view of prostaglandin E regulation of the immune response

Prostaglandins, particularly those of the E series, are widely regarded as immunosuppressive products of eukaryotic cells that can downregulate many aspects of B- and T-cell function. In this article, Richard Phipps and colleagues present a different concept of E series prostaglandins, based on recent evidence supporting a role for prostaglandins as potentiators of immunoglobulin class switching and of the synthesis of selected cytokines and cytokine receptors.

The maintenance and regulation of the humoral immune response: persisting antigen and the role of follicular antigen-binding dendritic cells as accessory cells.

The evidence indicating that antigen can persist for months or years in the spleens and draining lymph nodes of immunized animals is persuasive. This retained antigen Is almost exclusively, if not exclusively, associated with processes of dendritic cells in the follicles of these lymphoid organs (reviewed in this volume by Mandel et al. 1980), However, the role persisting antigen plays, once an immune response has been initiated, is open to question. It has been suggested that persisting antigen and specific antibody are the major components in an antibody feedback system which maintains and regulates antibody levels in vivo (Richter et al. 1965, Britton & MoUer 1968, Bystryn et at, 1971, Weigle 1975). It is well known that no additional antigen is required to maintain circulating antibody levels in hyperimmunized animals. Even when large volumes of blood are removed, antibody levels soon return to their previous levels. TTiis phenomenon might be explained by assuming that persisting antigen is unable to stimulate antibody synthesis

IL-8 Production in Human Lung Fibroblasts and Epithelial Cells Activated by the Pseudomonas Autoinducer N-3-Oxododecanoyl Homoserine Lactone Is Transcriptionally Regulated by NF-κB and Activator Protein-2

The destructive pulmonary inflammation associated with Pseudomonas aeruginosa colonization is caused, in part, by the production of the chemokine IL-8, which recruits neutrophils into the lung. The Pseudomonas autoinducer, N-3-oxododecanoyl homoserine lactone (3-O-C12-HSL), is a small lipid-soluble molecule that is essential in the regulation of many P. aeruginosa virulence factors, but little is known about how it affects eukaryotic cells. In this report we demonstrate that 3-O-C12-HSL is a potent stimulator of both IL-8 mRNA and protein from human fibroblasts and epithelial cells in vitro. The IL-8 produced from these 3-O-C12-HSL-stimulated cells was found to be functionally active by inducing the chemotaxis of neutrophils. To determine a mechanism for this IL-8 induction, deletion constructs of the IL-8 promoter were examined. It was found that the DNA region between nucleotides −1481 and −546 and the transcription factor NF-κB were essential for the maximal induction of IL-8 by 3-O-C12-HSL. This was confirmed by EMSAs, where 3-O-C12-HSL induced a shift with both AP-2 and NF-κB consensus DNA. The activation of NF-κB and subsequent production of IL-8 were found to be regulated by a mitogen-activated protein kinase pathway. These findings support the concept that the severe lung damage that accompanies P. aeruginosa infections is caused by an exuberant neutrophil response stimulated by 3-O-C12-HSL-induced IL-8. Understanding the mechanisms of 3-O-C12-HSL activation of lung structural cells may provide a means to help control lung damage during infections with P. aeruginosa.

Ultrafine particles and platelet activation in patients with coronary heart disease – results from a prospective panel study

BackgroundEpidemiological studies on health effects of air pollution have consistently shown adverse cardiovascular effects. Toxicological studies have provided evidence for thrombogenic effects of particles.A prospective panel study in a susceptible population was conducted in Erfurt, Germany, to study the effects of daily changes in ambient particles on various blood cells and soluble CD40ligand (sCD40L, also known as CD154), a marker for platelet activation that can cause increased coagulation and inflammation.Blood cells and plasma sCD40L levels were repeatedly measured in 57 male patients with coronary heart disease (CHD) during winter 2000/2001. Fixed effects linear regression models were applied, adjusting for trend, weekday and meteorological parameters.Hourly data on ultrafine particles (UFP, number concentration of particles from 0.01 to 0.1 μm), mass concentration of particles less than 10 and 2.5 μm in diameter (PM10, PM2.5), accumulation mode particle counts (AP, 0.1–1.0 μm), elemental and organic carbon, gaseous pollutants and meteorological data were collected at central monitoring sites.ResultsAn immediate increase in plasma sCD40L was found in association with UFP and AP (% change from geometric mean: 7.1; CI: [0.1, 14.5] and 6.9; CI: [0.5, 13.8], respectively). Platelet counts decreased in association with UFP showing an immediate, a three days delayed (lag 3) and a 5-day average response (% change from the mean: -1.8; CI: [-3.4,-0.2]; -2.4; CI: [-4.5,-0.3] and -2.2; CI: [-4.0,-0.3] respectively).ConclusionThe increased plasma sCD40L levels support the hypothesis that higher levels of ambient air pollution lead to an inflammatory response in patients with CHD thus providing a possible explanation for the observed association between air pollution and cardiovascular morbidity and mortality in susceptible parts of the population.

The nuclear receptor PPAR gamma is expressed by mouse T lymphocytes and PPAR gamma agonists induce apoptosis.

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.

Activation of human orbital fibroblasts through CD40 engagement results in a dramatic induction of hyaluronan synthesis and prostaglandin endoperoxide H synthase-2 expression: Insights into potential pathogenic mechanisms of thyroid-associated ophthalmopathy

Human orbital fibroblasts play a putative role in the pathogenesis of thyroid-associated ophthalmopathy (TAO). We hypothesize that the hyaluronan accumulation and inflammation in TAO derive from enhanced biosynthetic activities of orbital fibroblasts. CD40, a member of the tumor necrosis factor-α receptor superfamily, is a critical signaling molecule expressed by B lymphocytes. Engagement of CD40 with CD154 or CD40 ligand results in the activation of target genes. Orbital fibroblasts also display CD40. Here we report that CD40 engagement leads to substantial increases in hyaluronan synthesis in orbital fibroblasts. The increase is approximately 5-fold above control values, is comparable to the induction elicited by IL-1β and could be attenuated with dexamethasone but not by SC 58125, a prostaglandin endoperoxide H synthase-2 (PGHS-2)-selective inhibitor. PGHS-2 is also induced by CD40 engagement in a time-dependent manner, and this is mediated through increases in levels of steady-state mRNA. The induction of PGHS-2 leads to a dramatically enhanced prostaglandin E2 production that can be blocked by SC 58125 and dexamethasone. CD40 ligand up-regulates the synthesis of IL-1α, and blocking this cytokine with exogenous IL-1 receptor antagonist (IL-1ra) or with IL-1α neutralizing antibodies partially attenuates the induction of PGHS-2. In contrast, CD40 ligand up-regulation of hyaluronan synthesis is unaffected by IL-1ra. CD40 cross-linking enhances mitogen-activated protein kinase activation, and interrupting this pathway attenuates the PGHS-2 induction. Thus the CD40/CD40 ligand bridge represents a potentially important activational pathway for orbital fibroblasts that may underlie the cross-talk between these cells and leukocytes. These findings may be relevant to the pathogenesis of TAO and provide insights into previously unrecognized, potential therapeutic targets.

Peroxisome proliferator activator receptor-gamma agonists and 15-deoxy-Delta(12,14)(12,14)-PGJ(2) induce apoptosis in normal and malignant B-lineage cells.

The research described herein evaluates the expression and functional significance of peroxisome proliferator activator receptor-γ (PPAR-γ) on B-lineage cells. Normal mouse B cells and a variety of B lymphoma cells reflective of stages of B cell differentiation (e.g., 70Z/3, CH31, WEHI-231, CH12, and J558) express PPAR-γ mRNA and, by Western blot analysis, the 67-kDa PPAR-γ protein. 15-Deoxy-Δ12,14-PGJ2 (15d-PGJ2), a PPAR-γ agonist, has a dose-dependent antiproliferative and cytotoxic effect on normal and malignant B cells as shown by [3H]thymidine and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays. Only PPAR-γ agonists (thiazolidinediones), and not PPAR-α agonists, mimicked the effect of 15d-PGJ2 on B-lineage cells, indicating that the mechanism by which 15d-PGJ2 negatively affects B-lineage cells involves in part PPAR-γ. The mechanism by which PPAR-γ agonists induce cytotoxicity is via apoptosis, as shown by annexin V staining and as confirmed by DNA fragmentation detected using the TUNEL assay. Interestingly, addition of PGF2α, which was not known to affect lymphocytes, dramatically attenuated the deleterious effects of PPAR-γ agonists on B lymphomas. Surprisingly, 15d-PGJ2 induced a massive increase in nuclear mitogen-activated protein kinase activation, and pretreatment with PGF2α blunted the mitogen-activated protein kinase activation. This is the first study evaluating PPAR-γ expression and its significance on B lymphocytes. PPAR-γ agonists may serve as a counterbalance to the stimulating effects of other PGs, namely PGE2, which promotes B cell differentiation. Finally, the use of PGs, such as 15d-PGJ2, and synthetic PPAR-γ agonists to induce apoptosis in B-lineage cells may lead to the development of novel therapies for fatal B lymphomas.

The Pseudomonas Autoinducer N-(3-Oxododecanoyl) Homoserine Lactone Induces Cyclooxygenase-2 and Prostaglandin E2 Production in Human Lung Fibroblasts: Implications for Inflammation

Pseudomonas aeruginosa causes lethal lung infections in immunocompromised individuals such as those with cystic fibrosis. The lethality of these infections is directly associated with inflammation and lung tissue destruction. P. aeruginosa produces several acylated homoserine lactones (AHL) that are important in the regulation of bacterial virulence factors. Little is known about the effects of AHLs on human cells. In this work we report that the AHL N-(3-oxododecanoyl) homoserine lactone (3O-C12-HSL) from P. aeruginosa induces cyclooxygenase (Cox)-2, a seminal proinflammatory enzyme. When primary normal human lung fibroblasts were exposed to 3O-C12-HSL, an 8-fold induction in mRNA and a 35-fold increase in protein for Cox-2 were observed. In contrast, there was no substantial change in the expression of Cox-1. We also demonstrated that the induction of Cox-2 was regulated by 3O-C12-HSL activation of the transcription factor NF-κB. 3O-C12-HSL also stimulated an increase in the newly discovered inducible membrane-associated PGE synthase but had no effect on the expression of the cytosolic PGE synthase. We also demonstrate that 3O-C12-HSL stimulated the production of PGE2. PGE2 is known to induce mucus secretion, vasodilation, and edema, and acts as an immunomodulatory lipid mediator. We propose that 3O-C12-HSL induction of Cox-2, membrane-associated PGE synthase, and PGE2 likely contributes to the inflammation and lung pathology induced by P. aeruginosa infections in the lung. These studies further reinforce the concept that bacterial AHLs not only regulate bacterial virulence but also stimulate the activities of eukaryotic cells important for inflammation and immune defenses.

CD40 Expression by human fibroblasts

The purpose of this study was to determine whether human fibroblasts express CD40, a 50-kDa member of the tumor necrosis factor-alpha-receptor superfamily. CD40 is an important mitogenic receptor on B lymphocytes which regulates B lymphocyte proliferation and differentiation. Interestingly, CD40 mRNA was detected in human lung, gingival, synovial, dermal (foreskin), and spleen fibroblasts using the reverse-transcriptase polymerase chain reaction. Moreover, the CD40 protein was detected on cultured human fibroblasts using anti-CD40 mAbs (G28-5, EA-5) and flow cytometry and on fibroblasts in dermal tissue sections via in situ staining. In contrast to B lymphocytes, where CD40 expression is unregulated both by interleukin-4 and interferon (IFN-gamma), CD40 expression on cultured human fibroblasts could only be upregulated by IFN-gamma. IFN-gamma induced a 10-fold increase in CD40 mRNA and protein levels. Furthermore, via a two-color staining technique for CD40 expression and DNA content, IFN-gamma not only upregulated CD40 expression on cultured human fibroblasts, but also shifted fibroblasts into the G0/G1 phase of the cell cycle. This observation suggested that nonproliferating fibroblasts might display elevated levels of CD40. To test this hypothesis, CD40 expression was analyzed on fibroblasts in log phase growth vs fibroblasts which had reached confluency and were nonproliferating. Interestingly, confluent fibroblasts expressed higher levels of CD40 than fibroblasts in log phase growth. These data suggest that CD40 expression by human fibroblasts is related to cell growth. In summary, this report is the first to demonstrate that human fibroblasts from a variety of tissues display CD40. While the function of CD40 on fibroblasts is not yet known, it may facilitate fibroblast proliferation, an event important for tissue repair, and may facilitate inflammation via interaction with T lymphocytes and mast cells, which display the CD40 ligand.