Analyses of Calcium-Independent Phospholipase A2beta (iPLA2ß) in Biological Systems.

The Ca2+-independent phospholipases A2 (iPLA2s) are part of a diverse family of PLA2s, manifest activity in the absence of Ca2+, are ubiquitous, and participate in a variety of biological processes. Among the iPLA2s, the cytosolic iPLA2ß has received considerable attention and ongoing studies from various laboratories suggest that dysregulation of iPLA2ß can have a profound impact on the onset and/or progression of many diseases (e.g., cardiovascular, neurological, metabolic, autoimmune). Therefore, appropriate approaches are warranted to gain a better understanding of the role of iPLA2ß in vivo and its contribution to pathophysiology. Given that iPLA2ß is very labile, its basal expression is low in a number of cell systems, and that crystal structure of iPLA2ß is not yet available, careful and efficient protocols are needed to appropriately assess iPLA2ß biochemistry, dynamics, and membrane association. Here, step-by-step details are provided to (a) measure iPLA2ß-specific activity in cell lines or tissue preparations (using a simple radiolabel-based assay) and assess the impact of stimuli and inhibitors on resting- and disease-state iPLA2ß activity, (b) purify the iPLA2ß to near homogeneity (via sequential chromatography) from cell line or tissue preparations, enabling concentration of the enzyme for subsequent analyses (e.g., proteomics), and (c) employ hydrogen/deuterium exchange mass spectrometry analyses to probe both the structure of iPLA2ß and dynamics of its association with the membranes, substrates, and inhibitors.

A link between endoplasmic reticulum stress-induced ß-cell apoptosis and the group VIA Ca2+-independent phospholipase A2 (iPLA2ß).

Endoplasmic reticulum (ER) stress is becoming recognized as an important contributing factor in various diseases, including diabetes mellitus. Prolonged ER stress can cause ß-cell apoptosis; however, the underlying mechanism(s) that contribute to this process are not well understood. Early reports suggested that arachidonic acid metabolites and a Ca(2+)-independent phospholipase A(2) (iPLA(2)) activity play a role in ß-cell apoptosis. The PLA(2) family of enzymes catalyse the hydrolysis of the sn-2 substituent (i.e. arachidonic acid) of membrane phospholipids. In light of our findings that the pancreatic islet ß-cells are enriched in arachidonate-containing phospholipids and express the group VIA iPLA(2)ß, we considered the possibility that iPLA(2)ß participates in ER stress-induced ß-cell apoptosis. Our work revealed a novel mechanism, involving ceramide generation and triggering of mitochondrial abnormalities, by which iPLA(2)ß participates in the ß-cell apoptosis process. Here, we review our evidence linking ER stress, ß-cell apoptosis and iPLA(2)ß. Continued studies in this area will increase our understanding of the contribution of iPLA(2)ß to the evolution of diabetes mellitus and will further our knowledge of factors that influence ß-cell health in diabetes mellitus and identify potential targets for future therapeutic interventions to prevent ß-cell death.

Anti-phosphorylcholine-opsonized low-density lipoprotein promotes rapid production of proinflammatory cytokines by dendritic cells and natural killer cells.

BACKGROUND AND OBJECTIVE: Epidemiological and animal studies suggest that periodontal infections increase atherosclerosis risk. Periodontitis patients have elevated levels of anti-phosphorylcholine (anti-PC) reactive not only with numerous periodontal organisms but also with minimally modified low-density lipoprotein (mmLDL). Dendritic cells (DCs) reside in arterial walls and accumulate in atherosclerotic lesions. The ability of anti-PC to bind mmLDL prompted the hypothesis that opsonized mmLDL would stimulate DCs and enhance the production of proinflammatory cytokines that promote atherogenic plaque development. MATERIAL AND METHODS: Monocyte-derived DCs (mDCs) were generated using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, then stimulated with mmLDL or with anti-PC-opsonized mmLDL. The anti-PC effect was determined using flow cytometry, cofocal microscopy and cytokine assays. The production of CD83, IL-12p35 mRNA, IL-12p40 mRNA, IL-12p70 and IL-10 by DCs was monitored. RESULTS: Dendritic cells stimulated with mmLDL expressed little CD83 and produced little IL-12p70. However, anti-PC-opsonized mmLDL enhanced DC maturation, as indicated by upregulated CD83 and rapid (≤ 48 h) production of IL-12p70 if a source of interferon-γ (IFN-γ) was available. In leukocyte cultures, natural killer (NK) cells rapidly produced IFN-γ (≤ 48 h) when interacting with IL-12-producing DCs activated by anti-PC-opsonized mmLDL. Moreover, IFN-γ promoted DC IL-12 responses that were further augmented when mmLDL was opsonized with anti-PC. CONCLUSION: Minimally modified LDL-stimulated DCs and NK cells were mutually stimulatory, with DC IL-12p70 needed by NK cells and with NK cell IFN-γ needed by DCs. Moreover, production of these proinflammatory cytokines was markedly enhanced when LDL was opsonized by anti-PC. In short, the data suggest that the elevated anti-PC levels in periodontitis patients could promote a mechanism that facilitates atherosclerosis.

Group VI phospholipases A2: homeostatic phospholipases with significant potential as targets for novel therapeutics.

Group VI phospholipase A2 (PLA2) is a family of acyl hydrolases that targets the sn-2 fatty acid on the glycerophospholipid (GPL) backbone. These enzymes are grouped together based on structural homologies and catalytic activities that are independent of calcium and hence are also called the iPLA(2)s. Although the best characterized of these enzymes, iPLA2beta and iPLA2gamma, have long been proposed as homeostatic enzymes involved in basal GPL metabolism, recent studies indicate roles for these enzymes in biomedically relevant processes as well. For example, iPLA2 modulates calcium homeostasis by promoting replenishment of intracellular calcium stores. This function is likely of importance in the pathogenesis of Duchenne muscular dystrophy and potentially allergy as well. iPLA2 has a variety of roles in bacterial pathogenesis and the host response against bacterial and fungal infections. These characteristics suggest that the enzyme as a potential target to control infectious diseases. iPLA2 is linked to both proliferation and chemotherapy-induced apoptosis of tumor cells. As such, the enzyme is a potential target for cancer chemotherapy. Recent studies indicate essential roles for iPLA2 in glucose homeostasis, maintenance of energy balance, adipocyte development, and hepatic lipogenesis. Thus, the enzyme is an attractive target for drugs to control type II diabetes, fatty liver disease, and other manifestations of the metabolic syndrome. Several recent studies have associated iPLA2 inactivation with neurodegenerative diseases, suggesting the possibility that products of the iPLA2 reaction as potential treatments for these disorders. Together, these observations suggest iPLA2 as a novel and important target for drug development. However given the ubiquitous expression of the enzyme and its roles in basal GPL metabolism, drug strategies targeting iPLA2 must exhibit exquisite selectivity to avoid undesired side effects. Furthermore, the cell-specific nature of many iPLA2 functions may present another challenge in the design and implementation of drugs targeted to the enzyme.

Atherogenic lipoprotein parameters in patients with aggressive periodontitis.

BACKGROUND AND OBJECTIVE: Certain types of chronic infection increase the plasma level of very-low-density lipoprotein, leading to formation of the particularly atherogenic low-density lipoprotein subclass, small dense low-density lipoprotein. In the present study, we examined whether aggressive forms of periodontitis are associated with these atherogenic lipoprotein parameters. MATERIAL AND METHODS: Twelve healthy control subjects without periodontitis, 12 subjects with localized aggressive periodontitis and 12 subjects with generalized aggressive periodontitis were studied. Lipoprotein subclass levels were determined using nuclear magnetic resonance methodology. RESULTS: Healthy control subjects, localized aggressive periodontitis subjects and generalized aggressive periodontitis subjects had progressively higher plasma levels of very-low-density lipoprotein and progressively smaller average low-density lipoprotein size (p < 0.05, one-way analysis of variance). In pairwise comparisons, differences were only significant between healthy controls and generalized aggressive periodontitis subjects (p < 0.05, Tukey's post test). After adjustment for body mass index, the mean periodontal pocket depth correlated positively with plasma very-low-density lipoprotein levels (p = 0.047). Very-low-density lipoprotein concentrations correlated positively with small dense low-density lipoprotein levels and negatively with average low-density lipoprotein size. Prevalence of the atherogenic lipoprotein pattern-B in healthy controls, localized aggressive periodontitis subjects and generalized aggressive periodontitis subjects was 8.3%, 33.3% and 66.6%, respectively. CONCLUSION: These results indicate that periodontal infection is associated with elevated plasma levels of atherogenic lipoprotein species. This association may account for the increased risk of periodontitis patients for cardiovascular disease.

Differential platelet-activating factor synthesis by monocytes and polymorphonuclear leukocytes from subjects with localized aggressive periodontitis.

BACKGROUND AND OBJECTIVE: Platelet-activating factor is elevated in localized aggressive periodontitis. We previously demonstrated that the elevated level of platelet-activating factor in localized aggressive periodontitis is at least partially attributable to low levels of platelet-activating factor acetylhydrolase, the enzyme that catabolizes platelet-activating factor. The objective of this study was to determine if platelet-activating factor synthesis was also elevated in localized aggressive periodontitis. To test this, platelet-activating factor synthesis was quantified in the monocytes and polymorphonuclear neutrophils of periodontally healthy patients and of subjects with localized aggressive periodontitis. MATERIAL AND METHODS: Cells were labeled with [(3)H]acetate and treated with vehicle or stimulated with calcium ionophore A23187. Platelet-activating factor was extracted and quantified by scintillation counting. RESULTS: For both subject groups, resting monocytes and polymorphonuclear neutrophils produced platelet-activating factor, and calcium ionophore A23187 stimulated platelet-activating factor production in both cell types. However, calcium ionophore A23187-activated monocytes from subjects with localized aggressive periodontitis produced less platelet-activating factor than did activated periodontally healthy monocytes (p < 0.0001), suggesting an aberrant calcium ionophore A23187 response in monocytes from subjects with localized aggressive periodontitis. Indeed, when the data were expressed as fold induction of platelet-activating factor synthesis in response to calcium ionophore A23187, monocytes from subjects with localized aggressive periodontitis exhibited only a fourfold increase in platelet-activating factor synthesis, whereas calcium ionophore A23187-stimulated monocytes from periodontally healthy, chronic periodontitis and generalized aggressive periodontitis subjects produced approximately 12 times more platelet-activating factor than did resting monocytes. In contrast, both resting and activated localized aggressive periodontitis polymorphonuclear neutrophils synthesized more platelet-activating factor than did periodontally healthy polymorphonuclear neutrophils. CONCLUSION: These data suggest that high levels of platelet-activating factor in subjects with localized aggressive periodontitis result from both increased synthesis and reduced catabolism. While localized aggressive periodontitis polymorphonuclear neutrophils contribute to increased platelet-activating factor mass through synthesis, the contribution of monocytes is probably the result of reduced catabolism by platelet-activating factor acetylhydrolase.

Influence of proinflammatory cytokines on Actinobacillus actinomycetemcomitans specific IgG responses.

OBJECTIVE: High levels of serum anti-Actinobacillus actinomycetemcomitans immunoglobulin G (IgG) correlate with reduced extent and severity of periodontal disease and the present study was undertaken to begin testing the hypothesis that proinflammatory cytokines are important in the induction of optimal anti-A. actinomycetemcomitans IgG responses. BACKGROUND: Studies with pokeweed mitogen indicate that interleukin-1alpha (IL-1alpha) and IL-1beta are necessary for optimal IgG1 and IgG2 production and that prostaglandin E(2) (PGE(2)) and interferon-gamma (IFN-gamma) selectively promote IgG2, which is a major component of the anti-A. actinomycetemcomitans response in vivo. The pokeweed mitogen results suggest that these proinflammatory cytokines would also be necessary for optimal production of IgG specific for A. actinomycetemcomitans. METHODS: Peripheral blood mononuclear cells from A. actinomycetemcomitans-seropositive subjects with localized aggressive periodontitis were stimulated with A. actinomycetemcomitans in immune complexes capable of binding follicular dendritic cells that participate in the induction of recall responses in vivo. Cultures were manipulated with anti-IL-1alpha, anti-IL-1beta, anti-IFN-gamma, anti-IL-12, anti-CD21, indomethacin, and PGE(2). Actinobacillus actinomycetemcomitans specific IgG production was monitored by enzyme-linked immunosorbent assay (ELISA). RESULTS: Addition of follicular dendritic cells to peripheral blood mononuclear cells cultures resulted in follicular dendritic cell-lymphocyte clusters and increased anti-A. actinomycetemcomitans IgG responses (3-40-fold increases) compared with controls lacking follicular dendritic cells. Anti-IL-1alpha, anti-IL-1beta, anti-IFN-gamma, anti-IL-12, anti-CD21 and indomethacin suppressed anti-A. actinomycetemcomitans IgG production by half or more. PGE(2) restored IgG responses suppressed by indomethacin. CONCLUSIONS: The cytokines IL-1alpha, IL-1beta, IFN-gamma, IL-12, and PGE(2) were all necessary for optimal production of human anti-A. actinomycetemcomitans and the need for proinflammatory cytokines including the T helper 1 (Th1) cytokines is consistent with a response with a significant IgG2 component.

Dendritic-NK cell interactions in P. gingivalis-specific responses.

Patients with localized aggressive periodontitis have type-1 cytokines in gingival crevicular fluid and high titers of IFN-gamma-dependent IgG2 reactive with P. gingivalis in gingival crevicular fluid and serum. Localized aggressive periodontitis monocytes spontaneously differentiate into dendritic cells that can stimulate IFN-gamma production by NK cells. These relationships prompted the hypothesis that P. gingivalis-dendritic cell-NK cell interactions might promote type-1 cytokine responses. Although P. gingivalis is not a potent inducer of Th1 responses, it stimulated strong IL-12 responses by monocyte-derived dendritic cells in the presence of IFN-gamma, and IFN-gamma was produced by NK cells within 24 hrs in the presence of dendritic cells. Anti-P. gingivalis IgG2 responses were enhanced by dendritic cells, and removal of NK cells reduced IFN-gamma- and P. gingivalis-specific IgG2. Thus, P. gingivalis-dendritic cell-NK cell interactions apparently resulted in reciprocal stimulation and increased type-1 cytokine production by both dendritic cells and NK cells, and increased P. gingivalis-specific IgG2.

Dendritic cells stimulated with Actinobacillus actinomycetemcomitans elicit rapid gamma interferon responses by natural killer cells.

Human immunoglobulin G2 (IgG2) responses are gamma interferon (IFN-gamma) dependent, and monocyte-derived dendritic cells (mDCs) promote IgG2 production. DCs spontaneously emerge from monocytes in cultures prepared from localized aggressive periodontitis (LagP) patients, and these patients have high levels of IgG2 that is reactive with Actinobacillus actinomycetemcomitans. These results prompted the hypothesis that an interaction between mDCs and A. actinomycetemcomitans promotes IFN-gamma production, and IFN-gamma is known to promote both immunopathology and protective IgG2. A. actinomycetemcomitans induced mDCs to produce interleukin-12 (IL-12), and the addition of A. actinomycetemcomitans and DCs to cultured peripheral blood lymphocytes elicited high levels of IFN-gamma within just 24 h. In contrast, IL-4 was not detectable although DC-derived IL-10 production was apparent. A. actinomycetemcomitans-stimulated macrophages prepared from the same monocytes lacked the ability to induce IL-12 or IFN-gamma responses. NK cells of the innate immune system were the primary source of this early IFN-gamma, although CD8 T cells also contributed some. The NK cell-derived IFN-gamma was IL-12 dependent, and A. actinomycetemcomitans-DC interactions were Toll-like receptor 4 dependent. A. actinomycetemcomitans and A. actinomycetemcomitans lipopolysaccharide (LPS) were more potent than Escherichia coli and E. coli LPS in the ability to induce DC IL-12 and IFN-gamma. The ability of A. actinomycetemcomitans-stimulated DCs to induce NK cells to rapidly produce IFN-gamma in the absence of detectable IL-4 suggests their potential for skewing responses toward Th1. This may help explain the presence of Th1-associated cytokines in gingival crevicular fluid (GCF) from LagP patients and the high levels of IgG2 in their serum and GCF that is reactive with A. actinomycetemcomitans.

Anti-cardiolipin antibodies in sera from patients with periodontitis.

Antiphospholipid antibodies are commonly found in patients with systemic lupus erythematosus or the antiphospholipid syndrome, and a subset of such antibodies is associated with prothrombotic events such as stroke and with adverse pregnancy outcomes and fetal loss. We examined sera from 411 patients who were clinically characterized as to their periodontal disease status for serum levels of beta2-glycoprotein I-dependent anti-cardiolipin autoantibodies (anti-CL). The prevalence of patients with chronic periodontitis (CP) and generalized aggressive periodontitis (GAgP) positive for anti-CL (16.2% and 19.3%, respectively) was greater than that in healthy controls (NP) and localized aggressive periodontitis (LAgP) patients (6.8% and 3.2%). Patients with these autoantibodies demonstrated increased pocket depth and attachment loss compared with patients lacking the antibodies. Analysis of the data indicates that patients with generalized periodontitis have elevated levels of autoantibodies reactive with phospholipids. These antibodies could be involved in elevated risk for stroke, atherosclerosis, or pre-term birth in periodontitis patients.

Non-redundant roles for interleukin-1 alpha and interleukin-1 beta in regulating human IgG2.

BACKGROUND: Serum concentrations of immunoglobulin G2 (IgG2) are elevated in localized aggressive periodontitis (LAgP) patients, and secretory products of monocytes from LAgP patients enhance IgG2 responses of lymphocytes from healthy subjects. Furthermore, genes regulating production of interleukin (IL)-1 influence the risk for both aggressive periodontitis (AgP) and chronic periodontitis. These observations, and the fact that IgG2 dominates responses to carbohydrates from Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis, prompted the hypothesis that IL-1 alpha, IL-1 beta, and IL-RA may help regulate human IgG2 responses. METHODS: Human peripheral blood leukocytes (PBL) were stimulated in culture with pokeweed mitogen (PWM); the levels of available IL-1 gene products were manipulated; and the effect on IgG2 production was monitored. Manipulations of IL-1 were accomplished by adding specific neutralizing monoclonal antibodies or recombinant IL-1RA, IL-1 alpha, or IL-1 beta. RESULTS: Blocking the IL-1 receptor with IL-1RA or neutralizing IL-1 alpha or IL-1 beta with specific antibody dramatically suppressed IgG2 production (50% to 70%). Additionally IL-1 alpha did not compensate for neutralized IL-1 beta, and additional IL-1 beta did not compensate for neutralized IL-1 alpha, suggesting the 2 monokines have separate roles in promoting IgG2. Furthermore, combinations of anti-IL-1 alpha and anti-IL-1 beta were more inhibitory than either antibody alone, and IL-1 alpha and IL-1 beta in combination appeared to work additively in promoting IgG2. Moreover, PBL cultures from a group of LAgP patients with high IgG2 levels had elevated levels of IL-1 beta. CONCLUSION: IL-1 alpha and IL-1 beta appear to have critical and non-redundant roles in the generation and regulation of potent IgG2 responses, which appear to be important in human responses to carbohydrate-bearing bacteria.

Naegleria fowleri amoebae express a membrane-associated calcium-independent phospholipase A(2).

Naegleria fowleri, a free-living amoeba, is the causative agent of primary amoebic meningoencephalitis. Previous reports have demonstrated that N. fowleri expresses one or more forms of phospholipase A(2) (PLA(2)) and that a secreted form of this enzyme is involved in pathogenesis. However, the molecular nature of these phospholipases remains largely unknown. This study was initiated to determine whether N. fowleri expresses analogs of the well-characterized PLA(2)s that are expressed by mammalian macrophages. Amoeba cell homogenates contain a PLA(2) activity that hydrolyzes the substrate that is preferred by the 85 kDa calcium-dependent cytosolic PLA(2), cPLA(2). However, unlike the cPLA(2) enzyme in macrophages, this activity is largely calcium-independent, is constitutively associated with membranes and shows only a modest preference for phospholipids that contain arachidonate. The amoeba PLA(2) activity is sensitive to inhibitors that block the activities of cPLA(2)-alpha and the 80 kDa calcium-independent PLA(2), iPLA(2), that are expressed by mammalian cells. One of these compounds, methylarachidonyl fluorophosphonate, partially inhibits the constitutive release of [(3)H]arachidonic acid from pre-labeled amoebae. Together, these data suggest that N. fowleri expresses a constitutively active calcium-independent PLA(2) that may play a role in the basal phospholipid metabolism of these cells.

Invasion of human vascular endothelial cells by Actinobacillus actinomycetemcomitans via the receptor for platelet-activating factor.

Strains of the periodontal pathogen Actinobacillus actinomycetemcomitans are variable with respect to display of phosphorylcholine (PC)-bearing antigens. We have examined strains of A. actinomycetemcomitans with and without PC to assess their ability to invade endothelial cells via the receptor for platelet-activating factor (PAF). Results of antibiotic protection assays indicate that PC-bearing A. actinomycetemcomitans invade human vascular endothelial cells by a mechanism inhibitable by CV3988, a PAF receptor antagonist, and by PAF itself. The invasive phenotype was verified by transmission electron microscopy. A PC-deficient strain of this organism was not invasive. This property, in addition to the established ability of A. actinomycetemcomitans to invade epithelial cells, may provide this organism with access to the systemic circulation. The ability of PC-bearing oral bacteria to access the circulation may also explain the elevated levels of anti-PC antibody in serum found in patients with periodontitis.

Regulation of immunoglobulin G2 production by prostaglandin E(2) and platelet-activating factor.

Patients with localized juvenile periodontitis (LJP) have elevated levels of immunoglobulin G2 (IgG2) in their sera. This is also observed in vitro when peripheral blood leukocytes from LJP patients are stimulated with pokeweed mitogen. In previous studies, we showed that lymphocytes from subjects with no periodontitis (NP subjects) produced substantial amounts of IgG2 when they were cultured with monocytes from LJP patients (LJP monocytes). These observations indicate that monocytes or monocyte-derived mediators are positive regulators of the production of IgG2. The present study was initiated to determine if secreted factors from LJP monocytes were capable of enhancing IgG2 production and to determine if prostaglandin E2 (PGE(2)), which LJP monocytes produce at elevated levels, enhances IgG2 production. Experiments in a transwell system and with monocyte-conditioned media indicated that cell-cell contact was not necessary for LJP monocytes to augment the production of IgG2 by T and B cells from NP subjects. Moreover, the production of IgG2 was selectively induced by the addition of PGE(2) or platelet-activating factor (PAF), another lipid cytokine, which can elevate PGE(2) synthesis. Furthermore, IgG2 production was abrogated when cells were treated with indomethacin, a cyclooxygenase inhibitor that blocks the synthesis of PGE(2), or the PAF antagonists CV3988 and TEPC-15. The effects of indomethacin were completely reversed by PGE(2), indicating that this is the only prostanoid that is essential for the production of IgG2. Similarly, PGE(2) reversed the effects of a PAF antagonist, suggesting that the effects of PAF are mediated through the induction of PGE(2) synthesis. Together, these data indicate that PGE(2) and PAF are essential for the production of IgG2.

Regulation of phosphatidylcholine homeostasis by calcium-independent phospholipase A2.

Phosphatidylcholine (PtdCho) is the most abundant phospholipid in mammalian cell membranes and is essential for cell viability. The levels of this lipid must be tightly controlled to maintain homeostasis. Therefore, changes in the rate of PtdCho synthesis are generally balanced by changes in PtdCho catabolism and vice versa. It is commonly accepted that the rate of PtdCho synthesis is regulated by CTP:phosphocholine cytidylyltransferase (CT). However, it is not certain if PtdCho mass is regulated by specific catabolic enzyme(s). Our goal is to determine if PtdCho homeostasis is regulated by a phospholipase A2 (PLA2). To this end, we have prepared Chinese hamster ovary (CHO) cell lines that overexpress CT. CT activity is 7-10-fold higher in the transfected cells than in parental CHO cells. This increase in CT activity is associated with increases in both PtdCho synthesis and PtdCho catabolism. Glycerophosphocholine is the PtdCho catabolite that accumulates in the transfected cells, which suggests that PtdCho turnover is mediated by a phospholipase A2 (PLA2). Indeed, higher levels of calcium-independent PLA2 activity are measured in the cytosols of the CHO cells that overexpress CT, compared to parental CHO cells. The elevated calcium-independent PLA2 activity is associated with increases in the expression of the 80-kDa calcium-independent PLA2 (iPLA2). Together, these data suggest that the 80-kDa iPLA2 may be modulated in response to changes in PtdCho levels and therefore is involved in the regulation of PtdCho homeostasis in CHO cells.

Mature macrophage cell lines exhibit variable responses to LPS.

Bacterial lipopolysaccharide (LPS) is a potent activator of cells of the macrophage/monocyte lineage. Two mature macrophage cell lines, P388D1 and RAW264.7, exhibit very different biological responses to LPS. Although RAW264.7 cells release arachidonic acid from phospholipid in response to LPS stimulation, P388D1 cells do not respond in this manner. However, LPS primes P388D1 cells to release arachidonic acid in response to other stimuli. The goal of this work is to contrast the biochemical events that occur in LPS-treated P388D1 and RAW264.7 macrophages. Enzyme assays indicate that LPS treatment induces the activation of cytosolic PLA2 in RAW264.7, but not in P388D1 cells. Phorbol ester (PMA), a receptor-independent stimulus, also fails to induce arachidonic acid release from P388D1 cells, suggesting that these cells may have a defect in the signal transduction machinery that is common to LPS and PMA. This hypothesis is supported by the observation that the expression of the LPS receptors CD14 and CD11b/CD18 is similar on P388D1 and RAW264.7 cells. Western blot analyses indicate that the erk kinases are activated upon LPS treatment of RAW264.7 but not P388D1 cells. LPS-induced arachidonic acid release is reduced in cells treated with the MEK inhibitor PD98059, suggesting that activated erk kinases mediate the phosphorylation and activation of cPLA2 in this system. Interestingly, the p42 isoform of erk (erk2) appears to be activated in resting P388D1 cells. This observation indicates that the MAP kinase cascade may be constitutively activated in P388D1 cells which may in turn limit their ability to respond to LPS. Together, these data provide evidence that mature macrophages from different sources can exhibit variable responses to LPS and highlight the danger of making generalizations regarding the effects of LPS on macrophages.

Tobacco and smoking: environmental factors that modify the host response (immune system) and have an impact on periodontal health.

This review summarizes the current data on the effects of smoking and tobacco on the immune system and its potential impact on periodontal health. Smokers are 2.5-6 times more likely to develop periodontal disease than non-smokers, and there is evidence for a direct correlation between the number of cigarettes smoked and the risk of developing disease. Tobacco users also tend to exhibit increased severity of periodontal disease. Direct correlations between tobacco use and increased attachment loss and pocket depth and reduced bone crest height have been reported. Although the correlation between tobacco use and periodontal disease is quite strong, the role of tobacco in the pathogenesis of periodontal disease is uncertain. Recent studies indicate that one potential mechanism is that tobacco use exacerbates periodontal disease because it alters the immune response to periodontal pathogens. Indeed, smokers exhibit increased numbers of peripheral blood mononuclear phagocytes which appear to be functionally compromised. Inadequate phagocyte activity could reduce the clearance of pathogens from the oral cavity and thereby facilitate the development of periodontal disease. Tobacco-exposed B- and T-lymphocytes exhibit reduced proliferative capacities which could limit the production of protective immunoglobulins against oral pathogens. The risk factors for periodontal disease can be broadly classified as genetic, environmental, host-response factors, and host-related factors such as age. Tobacco, an environmental factor, undermines the host response and may facilitate the development and progression of periodontal disease. This review highlights the inter-relatedness of two of the risk factors associated with periodontal disease.

Coordinate regulation of stress- and mitogen-activated protein kinases in the apoptotic actions of ceramide and sphingosine.

We characterized participation of the stress-activated protein kinase (SAPK) cascade in the lethal actions of the cytotoxic lipid messengers ceramide and sphingosine in U937 human monoblastic leukemia cells. Acute exposure of U937 cells to either lipid resulted in loss of proliferative capacity, degradation of genomic DNA, and manifestation of apoptotic cytoarchitecture. Ceramide robustly stimulated p46-JNK1/p54-JNK2 activity and increased expression of c-jun mRNA and c-Jun protein; in contrast, sphingosine moderately stimulated p46-JNK1/p54-JNK2 and failed to modify c-jun/c-Jun expression. Dominant-negative blockade of normal c-Jun activity by transfection with the TAM-67 c-Jun NH2-terminal deletion mutant abolished the lethal actions of ceramide but was without effect on those of sphingosine, indicating that ceramide-related apoptosis is directly dependent on activation of c-Jun, whereas sphingosine-induced cell death proceeds via an unrelated downstream mechanism. Characterization of the mitogen-activated protein kinase (MAPK) cascade in these responses revealed a further functional disparity between the two lipids: basal p42-ERK1/ p44-ERK2 activity was gradually reduced by ceramide but immediately and completely suppressed by sphingosine. Moreover, blockade of the MAPK cascade by the aminomethoxyflavone MEK1 inhibitor PD-98059 unexpectedly activated p46-JNK1/p54-JNK2 and induced apoptosis in a manner qualitatively resembling that of sphingosine. Both lipids sharply increased p38-RK activity; selective pharmacological inhibition of p38-RK by the pyridinyl imidazole SB-203580 failed to mitigate the cytotoxicity associated with either ceramide or sphingosine, suggesting that p38-RK is not essential for lipid-induced apoptosis. These findings demonstrate that reciprocal alterations in the SAPK and MAPK cascades are associated with the apoptotic influence of either lipid inasmuch as (i) ceramide-mediated lethality is primarily associated with strong stimulation of SAPK and weak inhibition of MAPK, whereas (ii) sphingosine-mediated lethality is primarily associated with weak stimulation of SAPK and strong inhibition of MAPK. We therefore propose that leukemic cell survival depends on the maintenance of an imbalance of the outputs from the MAPK and SAPK systems such that the dominant basal influence of the MAPK cascade allows sustained proliferation, whereas acute redirection of this balance toward the SAPK cascade initiates apoptotic cell death.

Arachidonic acid mobilization in P388D1 macrophages is controlled by two distinct Ca(2+)-dependent phospholipase A2 enzymes.

Macrophage-like P388D1 cells mobilize arachidonic acid (AA) and produce prostaglandin E2 upon stimulation with bacterial lipopolysaccharide and platelet-activating factor. We have now demonstrated that AA mobilization in these cells is composed of two distinct events: a transient phase in which AA accumulates in the cell and a sustained phase in which the fatty acid accumulates in the incubation medium. Both phases are markedly dependent on the presence of Ca2+ in the extracellular medium. Treatment with an antisense oligonucleotide to group II phospholipase A2 inhibits the accumulation of AA in the incubation medium, but has no effect on the accumulation of this fatty acid in the cell. In addition, treatment with antisense oligonucleotide to group II phospholipase A2 has no effect on the uptake or the esterification of AA. Collectively, these results indicate that, in addition to the previously demonstrated role of group II phospholipase A2 in AA mobilization in activated P388D1 cells, another phospholipase A2, distinct from the group II enzyme, is implicated in raising the levels of intracellular AA during the early steps of P388D1 cell activation and in modulating deacylation/reacylation reactions involving AA. The data suggest that each of the different phospholipase A2 enzymes present in P388D1 cells serves a distinct role in cell function.

Antisense inhibition of group II phospholipase A2 expression blocks the production of prostaglandin E2 by P388D1 cells.

Macrophage-like P388D1 cells release [3H]arachidonic acid and produce prostaglandin E2 (PGE2) upon stimulation with bacterial lipopolysaccharide (LPS) and platelet-activating factor (PAF). To determine whether group II phospholipase A2 (PLA2) is involved in this release, we treated P388D1 cells with antisense inhibitors specific for group II PLA2 RNA. Treatment with oligonucleotide ASGII decreased PLA2 activity in P388D1 cell homogenates by approximately 60% and reduced the release of [3H]arachidonic acid and PGE2 from activated cells to nearly resting cell levels. The inhibition by antisense oligonucleotide ASGII was blocked when its sense complement, SGII, was included in the incubation mixture. Stably transfected P388D1 cells expressing an antisense construct for group II PLA2 also produced reduced quantities of PGE2 in response to LPS and PAF. These data suggest that prostaglandin production by activated P388D1 cells involves phospholipid hydrolysis by group II PLA2. Oligonucleotide ASGII also blocked the appearance of a heparin-releasable group II PLA2 in the culture supernatants of P388D1 cells. The disappearance of this protein correlated with reduced PGE2 production by activated cells, indicating that an extracellular heparin-associated pool of group II PLA2 is involved in prostaglandin production by P388D1 cells.