Alterations in ß-Cell Sphingolipid Profile Associated with ER Stress and iPLA2ß: Another Contributor to ß-Cell Apoptosis in Type 1 Diabetes.

Type 1 diabetes (T1D) development, in part, is due to ER stress-induced ß-cell apoptosis. Activation of the Ca2+-independent phospholipase A2 beta (iPLA2ß) leads to the generation of pro-inflammatory eicosanoids, which contribute to ß-cell death and T1D. ER stress induces iPLA2ß-mediated generation of pro-apoptotic ceramides via neutral sphingomyelinase (NSMase). To gain a better understanding of the impact of iPLA2ß on sphingolipids (SLs), we characterized their profile in ß-cells undergoing ER stress. ESI/MS/MS analyses followed by ANOVA/Student's t-test were used to assess differences in sphingolipids molecular species in Vector (V) control and iPLA2ß-overexpressing (OE) INS-1 and Akita (AK, spontaneous model of ER stress) and WT-littermate (AK-WT) ß-cells. As expected, iPLA2ß induction was greater in the OE and AK cells in comparison with V and WT cells. We report here that ER stress led to elevations in pro-apoptotic and decreases in pro-survival sphingolipids and that the inactivation of iPLA2ß restores the sphingolipid species toward those that promote cell survival. In view of our recent finding that the SL profile in macrophages-the initiators of autoimmune responses leading to T1D-is not significantly altered during T1D development, we posit that the iPLA2ß-mediated shift in the ß-cell sphingolipid profile is an important contributor to ß-cell death associated with T1D.

Macrophage polarization is linked to Ca2+-independent phospholipase A2ß-derived lipids and cross-cell signaling in mice.

Phospholipases A2 (PLA2s) catalyze hydrolysis of the sn-2 substituent from glycerophospholipids to yield a free fatty acid (i.e., arachidonic acid), which can be metabolized to pro- or anti-inflammatory eicosanoids. Macrophages modulate inflammatory responses and are affected by Ca2+-independent phospholipase A2 (PLA2)ß (iPLA2ß). Here, we assessed the link between iPLA2ß-derived lipids (iDLs) and macrophage polarization. Macrophages from WT and KO (iPLA2ß-/-) mice were classically M1 pro-inflammatory phenotype activated or alternatively M2 anti-inflammatory phenotype activated, and eicosanoid production was determined by ultra-performance LC ESI-MS/MS. As a genotypic control, we performed similar analyses on macrophages from RIP.iPLA2ß.Tg mice with selective iPLA2ß overexpression in ß-cells. Compared with WT, generation of select pro-inflammatory prostaglandins (PGs) was lower in iPLA2ß-/- , and that of a specialized pro-resolving lipid mediator (SPM), resolvin D2, was higher; both changes are consistent with the M2 phenotype. Conversely, macrophages from RIP.iPLA2ß.Tg mice exhibited an opposite landscape, one associated with the M1 phenotype: namely, increased production of pro-inflammatory eicosanoids (6-keto PGF1α, PGE2, leukotriene B4) and decreased ability to generate resolvin D2. These changes were not linked with secretory PLA2 or cytosolic PLA2α or with leakage of the transgene. Thus, we report previously unidentified links between select iPLA2ß-derived eicosanoids, an SPM, and macrophage polarization. Importantly, our findings reveal for the first time that ß-cell iPLA2ß-derived signaling can predispose macrophage responses. These findings suggest that iDLs play critical roles in macrophage polarization, and we posit that they could be targeted therapeutically to counter inflammation-based disorders.

Group VIA Phospholipase A2 (iPLA2ß) Modulates Bcl-x 5'-Splice Site Selection and Suppresses Anti-apoptotic Bcl-x(L) in ß-Cells.

Diabetes is a consequence of reduced ß-cell function and mass, due to ß-cell apoptosis. Endoplasmic reticulum (ER) stress is induced during ß-cell apoptosis due to various stimuli, and our work indicates that group VIA phospholipase A2ß (iPLA2ß) participates in this process. Delineation of underlying mechanism(s) reveals that ER stress reduces the anti-apoptotic Bcl-x(L) protein in INS-1 cells. The Bcl-x pre-mRNA undergoes alternative pre-mRNA splicing to generate Bcl-x(L) or Bcl-x(S) mature mRNA. We show that both thapsigargin-induced and spontaneous ER stress are associated with reductions in the ratio of Bcl-x(L)/Bcl-x(S) mRNA in INS-1 and islet ß-cells. However, chemical inactivation or knockdown of iPLA2ß augments the Bcl-x(L)/Bcl-x(S) ratio. Furthermore, the ratio is lower in islets from islet-specific RIP-iPLA2ß transgenic mice, whereas islets from global iPLA2ß(-/-) mice exhibit the opposite phenotype. In view of our earlier reports that iPLA2ß induces ceramide accumulation through neutral sphingomyelinase 2 and that ceramides shift the Bcl-x 5'-splice site (5'SS) selection in favor of Bcl-x(S), we investigated the potential link between Bcl-x splicing and the iPLA2ß/ceramide axis. Exogenous C6-ceramide did not alter Bcl-x 5'SS selection in INS-1 cells, and neutral sphingomyelinase 2 inactivation only partially prevented the ER stress-induced shift in Bcl-x splicing. In contrast, 5(S)-hydroxytetraenoic acid augmented the ratio of Bcl-x(L)/Bcl-x(S) by 15.5-fold. Taken together, these data indicate that ß-cell apoptosis is, in part, attributable to the modulation of 5'SS selection in the Bcl-x pre-mRNA by bioactive lipids modulated by iPLA2ß.

Role of calcium-independent phospholipase A(2)ß in human pancreatic islet ß-cell apoptosis.

Death of ß-cells due to apoptosis is an important contributor to ß-cell dysfunction in both type 1 and type 2 diabetes mellitus. Previously, we described participation of the Group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)ß) in apoptosis of insulinoma cells due to ER stress. To examine whether islet ß-cells are similarly susceptible to ER stress and undergo iPLA(2)ß-mediated apoptosis, we assessed the ER stress response in human pancreatic islets. Here, we report that the iPLA(2)ß protein is expressed predominantly in the ß-cells of human islets and that thapsigargin-induced ER stress promotes ß-cell apoptosis, as reflected by increases in activated caspase-3 in the ß-cells. Furthermore, we demonstrate that ER stress is associated with increases in islet iPLA(2)ß message, protein, and activity, iPLA(2)ß-dependent induction of neutral sphingomyelinase and ceramide accumulation, and subsequent loss of mitochondrial membrane potential. We also observe that basal activated caspase-3 increases with age, raising the possibility that ß-cells in older human subjects have a greater susceptibility to undergo apoptotic cell death. These findings reveal for the first time expression of iPLA(2)ß protein in human islet ß-cells and that induction of iPLA(2)ß during ER stress contributes to human islet ß-cell apoptosis. We hypothesize that modulation of iPLA(2)ß activity might reduce ß-cell apoptosis and this would be beneficial in delaying or preventing ß-cell dysfunction associated with diabetes.

Group VIA Ca2+-independent phospholipase A2 (iPLA2beta) and its role in beta-cell programmed cell death.

Activation of phospholipases A(2) (PLA(2)s) leads to the generation of biologically active lipid mediators that can affect numerous cellular events. The Group VIA Ca(2+)-independent PLA(2), designated iPLA(2)beta, is active in the absence of Ca(2+), activated by ATP, and inhibited by the bromoenol lactone suicide inhibitor (BEL). Over the past 10-15 years, studies using BEL have demonstrated that iPLA(2)beta participates in various biological processes and the recent availability of mice in which iPLA(2)beta expression levels have been genetically-modified are extending these findings. Work in our laboratory suggests that iPLA(2)beta activates a unique signaling cascade that promotes beta-cell apoptosis. This pathway involves iPLA(2)beta dependent induction of neutral sphingomyelinase, production of ceramide, and activation of the intrinsic pathway of apoptosis. There is a growing body of literature supporting beta-cell apoptosis as a major contributor to the loss of beta-cell mass associated with the onset and progression of Type 1 and Type 2 diabetes mellitus. This underscores a need to gain a better understanding of the molecular mechanisms underlying beta-cell apoptosis so that improved treatments can be developed to prevent or delay the onset and progression of diabetes mellitus. Herein, we offer a general review of Group VIA Ca(2+)-independent PLA(2) (iPLA(2)beta) followed by a more focused discussion of its participation in beta-cell apoptosis. We suggest that iPLA(2)beta-derived products trigger pathways which can lead to beta-cell apoptosis during the development of diabetes.

Spontaneous development of endoplasmic reticulum stress that can lead to diabetes mellitus is associated with higher calcium-independent phospholipase A2 expression: a role for regulation by SREBP-1.

Our recent studies indicate that endoplasmic reticulum (ER) stress causes INS-1 cell apoptosis by a Ca(2+)-independent phospholipase A(2) (iPLA(2)beta)-mediated mechanism that promotes ceramide generation via sphingomyelin hydrolysis and subsequent activation of the intrinsic pathway. To elucidate the association between iPLA(2)beta and ER stress, we compared beta-cell lines generated from wild type (WT) and Akita mice. The Akita mouse is a spontaneous model of ER stress that develops hyperglycemia/diabetes due to ER stress-induced beta-cell apoptosis. Consistent with a predisposition to developing ER stress, basal phosphorylated PERK and activated caspase-3 are higher in the Akita cells than WT cells. Interestingly, basal iPLA(2)beta, mature SREBP-1 (mSREBP-1), phosphorylated Akt, and neutral sphingomyelinase (NSMase) are higher, relative abundances of sphingomyelins are lower, and mitochondrial membrane potential (DeltaPsi) is compromised in Akita cells, in comparison with WT cells. Exposure to thapsigargin accelerates DeltaPsi loss and apoptosis of Akita cells and is associated with increases in iPLA(2)beta, mSREBP-1, and NSMase in both WT and Akita cells. Transfection of Akita cells with iPLA(2)beta small interfering RNA, however, suppresses NSMase message, DeltaPsi loss, and apoptosis. The iPLA(2)beta gene contains a sterol-regulatory element, and transfection with a dominant negative SREBP-1 reduces basal mSREBP-1 and iPLA(2)beta in the Akita cells and suppresses increases in mSREBP-1 and iPLA(2)beta due to thapsigargin. These findings suggest that ER stress leads to generation of mSREBP-1, which can bind to the sterol-regulatory element in the iPLA(2)beta gene to promote its transcription. Consistent with this, SREBP-1, iPLA(2)beta, and NSMase messages in Akita mouse islets are higher than in WT islets.

Calcium-independent phospholipase A2 participates in KCl-induced calcium sensitization of vascular smooth muscle.

In vascular smooth muscle, KCl not only elevates intracellular free Ca(2+) ([Ca(2+)](i)), myosin light chain kinase activity and tension (T), but also can inhibit myosin light chain phosphatase activity by activation of rhoA kinase (ROCK), resulting in Ca(2+) sensitization (increased T/[Ca(2+)](i) ratio). Precisely how KCl causes ROCK-dependent Ca(2+) sensitization remains to be determined. Using Fura-2-loaded isometric rings of rabbit artery, we found that the Ca(2+)-independent phospholipase A(2) (iPLA(2)) inhibitor, bromoenol lactone (BEL), reduced the KCl-induced tonic but not early phasic phase of T and potentiated [Ca(2+)](i), reducing Ca(2+) sensitization. The PKC inhibitor, GF-109203X (> or =3 microM) and the pseudo-substrate inhibitor of PKCzeta produced a response similar to BEL. BEL reduced basal and KCl-stimulated myosin phosphatase phosphorylation. Whereas BEL and H-1152 produced strong inhibition of KCl-induced tonic T (approximately 50%), H-1152 did not induce additional inhibition of tissues already inhibited by BEL, suggesting that iPLA(2) links KCl stimulation with ROCK activation. The cPLA(2) inhibitor, pyrrolidine-1, inhibited KCl-induced tonic increases in [Ca(2+)](i) but not T, whereas the inhibitor of 20-HETE production, HET0016, acted like the ROCK inhibitor H-1152 by causing Ca(2+) desensitization. These data support a model in which iPLA(2) activity regulates Ca(2+) sensitivity.

Apoptosis induces expression of sphingosine kinase 1 to release sphingosine-1-phosphate as a "come-and-get-me" signal.

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates myriad important cellular processes, including growth, survival, cytoskeleton rearrangements, motility, and immunity. Here we report that treatment of Jurkat and U937 leukemia cells with the pan-sphingosine kinase (SphK) inhibitor N,N-dimethylsphingosine to block S1P formation surprisingly caused a large increase in expression of SphK1 concomitant with induction of apoptosis. Another SphK inhibitor, D,L-threo-dihydrosphingosine, also induced apoptosis and produced dramatic increases in SphK1 expression. However, up-regulation of SphK1 was not a specific effect of its inhibition but rather was a consequence of apoptotic stress. The chemotherapeutic drug doxorubicin, a potent inducer of apoptosis in these cells, also stimulated SphK1 expression and activity and promoted S1P secretion. The caspase inhibitor ZVAD reduced not only doxorubicin-induced lethality but also the increased expression of SphK1 and secretion of S1P. Apoptotic cells secrete chemotactic factors to attract phagocytic cells, and we found that S1P potently stimulated chemotaxis of monocytic THP-1 and U937 cells and primary monocytes and macrophages. Collectively, our data suggest that apoptotic cells may up-regulate SphK1 to produce and secrete S1P that serves as a "come-and-get-me" signal for scavenger cells to engulf them in order to prevent necrosis.

Inhibition of calcium-independent phospholipase A2 suppresses proliferation and tumorigenicity of ovarian carcinoma cells.

PLA2 (phospholipase A2) enzymes play critical roles in membrane phospholipid homoeostasis and in generation of lysophospholipid growth factors. In the present study, we show that the activity of the cytosolic iPLA2 (calcium-independent PLA2), but not that of the calcium-dependent cPLA2 (cytosolic PLA2), is required for growth-factor-independent, autonomous replication of ovarian carcinoma cells. Blocking iPLA2 activity with the pharmacological inhibitor BEL (bromoenol lactone) induces cell cycle arrest in S- and G2/M-phases independently of the status of the p53 tumour suppressor. Inhibition of iPLA2 activity also leads to modest increases in apoptosis of ovarian cancer cells. The S- and G2/M-phase accumulation is accompanied by increased levels of the cell cycle regulators cyclins B and E. Interestingly, the S-phase arrest is released by supplementing the growth factors LPA (lysophosphatidic acid) or EGF (epidermal growth factor). However, inhibition of iPLA2 activity with BEL remains effective in repressing growth-factor- or serum-stimulated proliferation of ovarian cancer cells through G2/M-phase arrest. Down-regulation of iPLA2b expression with lentivirus-mediated RNA interference inhibited cell proliferation in culture and tumorigenicity of ovarian cancer cell lines in nude mice. These results indicate an essential role for iPLA2 in cell cycle progression and tumorigenesis of ovarian carcinoma cells.

The immunosuppressant drug FTY720 inhibits cytosolic phospholipase A2 independently of sphingosine-1-phosphate receptors.

FTY720 is a potent immunomodulator drug that inhibits the egress of lymphocytes from secondary lymphoid tissues and thymus. FTY720 is phosphorylated in vivo by sphingosine kinase 2 to FTY720-phosphate, which acts as a potent sphingosine-1-phosphate (S1P) receptor agonist. However, in contrast to S1P, FTY720 has no effect on mast-cell degranulation, yet significantly reduces antigen-induced secretion of PGD2 and cysteinyl-leukotriene. Unexpectedly, this effect of FTY720 was independent of its phosphorylation and S1P receptor functions. The rate-limiting step in the biosynthesis of all eicosanoids is the phospholipase A2 (PLA2)-mediated release of arachidonic acid from glycerol phospholipids. Although FTY720 also reduced arachidonic acid release in response to antigen, it had no effect on translocation of cPLA2 or ERK1/2 activation, suggesting that it does not interfere with FcepsilonRI-mediated events leading to cPLA2 activation. Remarkably, however, FTY720 drastically inhibited recombinant cPLA2alpha activity, whereas FTY720-phosphate, sphingosine, or S1P had no effect. This study has uncovered a unique action of FTY720 as an inhibitor of cPLA2alpha and hence on production of all eicosanoids. Our results have important implications for the potential therapeutic mechanism of action of FTY720 in eicosanoid-driven inflammatory disorders such as asthma and multiple sclerosis.

Altered lipoprotein subclass distribution and PAF-AH activity in subjects with generalized aggressive periodontitis.

In this study, we examined whether the documented increase of plasma triglycerides in patients with generalized aggressive periodontitis (GAgP) is associated with changes in lipoprotein subclass distribution and/or LDL-associated platelet-activating factor acetylhydrolase (PAF-AH) activity. Lipoprotein subclasses were analyzed in whole plasma samples using nuclear magnetic resonance methods. Compared with subjects without periodontitis (NP subjects; n = 12), GAgP subjects (n = 12) had higher plasma levels of large, medium, and small VLDL (35.0 +/- 6.7 vs. 63.1 +/- 9.6 nmol/l; P = 0.025), higher levels of intermediate density lipoprotein (24.8 +/- 11.6 vs. 87.2 +/- 16.6 nmol/l; P = 0.006), lower levels of large LDL (448.3 +/- 48.5 vs. 315.8 +/- 59.4 nmol/l; P = 0.098), and higher levels of small LDL (488.2 +/- 104.2 vs. 946.7 +/- 151.6 nmol/l; P = 0.021). The average size of LDL from NP and GAgP subjects was 21.4 +/- 0.2 and 20.6 +/- 0.3 nm, respectively (P = 0.031). Compared with NP subjects, GAgP subjects had a greater number of circulating LDL particles (961.3 +/- 105.3 vs. 1,349.0 +/- 133.2 nmol/l; P = 0.032). Differences in the plasma levels of large, medium, and small HDL were not statistically significant. NP and GAgP subjects had similar plasma levels of total LDL-associated PAF-AH activity; however, LDL of GAgP subjects contained less PAF-AH activity per microgram of LDL protein (1,458.0 +/- 171.0 and 865.2 +/- 134 pmol/min/microg; P = 0.014). These results indicate that, in general, GAgP subjects have a more atherogenic lipoprotein profile and lower LDL-associated PAF-AH activity than NP subjects. These differences may help explain the increased risk of GAgP subjects for cardiovascular disease.

Regulation of group VIA phospholipase A2 expression by sterol availability.

Several lines of evidence suggest that glycerophospholipid mass is maintained through the coordinate regulation of CTP:phosphocholine cytidylyltransferase-alpha (CTalpha) and the group VIA calcium-independent phospholipase A2 (iPLA2). CTalpha expression is modulated by sterol and this is mediated in part through sterol regulatory element binding proteins (SREBP). In this report, we investigate the possibility that iPLA2 expression is controlled in a similar manner. When Chinese hamster ovary (CHO) cells were cultured under sterol-depleted conditions, iPLA2 catalytic activity, mRNA, and protein were induced by between two- and threefold. These inductions were suppressed when the cells were supplemented with exogenous sterols. Luciferase reporter assays indicated that sterol depletion induced transcription of iPLA2, an analysis of the 5' flanking region suggested that the iPLA2 gene contained a putative sterol regulatory element (SRE), and electrophoretic mobility shift assay (EMSA) analysis indicated that this element can bind SREBP-2. Notably, a mutant CHO cell line (SRD4) that constitutively generates mature SREBP proteins exhibited increased iPLA2 activity and expression compared to wild-type cells. These data suggest that iPLA2 expression is regulated in a manner consistent with other important genes in sterol and glycerophospholipid metabolism. Such coordinate regulation may be essential for maintaining the lipid composition of cell membranes.

Cell cycle dependence of group VIA calcium-independent phospholipase A2 activity.

Homeostasis of phosphatidylcholine (PC) is regulated by the opposing actions between CTP:phosphocholine cytidylyltransferase (CT) and the group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)). We investigated this process during the cell cycle. PC mass doubles during late G(1) and early S phase when its rate of catabolism is lowest. We show that iPLA(2) activity is cell cycle-dependent with peak activity during G(2)/M and late S phase. iPLA(2) activity declines during G(1) and is lowest at the G(1)/S transition and early S phase. The accumulation of PC correlates with decreased iPLA(2) activity, suggesting that regulation of this enzyme contributes to phospholipid accumulation. The levels of 80 kDa iPLA(2) protein do not change and thus cannot account for changes in enzyme activity. Reverse transcriptase and real-time PCR experiments show that splice variant iPLA(2) mRNAs are preferentially expressed during G(2)/M. Immunoblot analyses with an antibody directed against the N terminus of iPLA(2) revealed a approximately 50 kDa protein that is of appropriate size to be the truncated protein encoded by the ankyrin-iPLA(2)-1 splice variant mRNA. The levels of truncated iPLA(2) protein were high in cells in late G(1) and S phase cells that had low iPLA(2) activity and low in G(2)/M cells that had high iPLA(2) activity. The truncated protein co-immunoprecipitated with full-length iPLA(2), indicating a physical interaction between the two proteins. Together, these data suggest that truncated iPLA(2) proteins associate with active iPLA(2) and down-regulate its activity during G(1). This down-regulation may contribute to phospholipid accumulation during the cell cycle.

Modulation of adaptive immune responses by sphingosine-1-phosphate.

Sphingosine-1-phosphate (S1P) has long been recognized as a mediator of a variety of cell functions. A growing body of evidence has accumulated demonstrating its role in cell migration and as a mediator of growth factor-induced events. In recent years, it has become apparent that S1P also mediates many cytokine and chemokine functions. Cells of the immune system function and migrate in response to a complex network of cytokines and chemokines, and the outcome is determined by the interplay of the effects of these molecules on the target cell. S1P may be a bona fide component of these networks and influence the responses of cells to these immune modulators.

Delineation of the role of platelet-activating factor in the immunoglobulin G2 antibody response.

Localized aggressive periodontitis (LAgP) is a chronic inflammatory disease characterized by severe destruction of periodontal tissues surrounding the first molars and incisors. LAgP subjects produce large amounts of immunoglobulin G2 (IgG2) antibody against oral pathogens, and this response is inversely correlated with the severity of disease. We previously demonstrated that platelet-activating factor (PAF) is required for optimal IgG2 responses. The present investigation was designed to determine the mechanism of IgG2 induction by PAF. Exogenous PAF acetylhydrolase suppressed approximately 80% of pokeweed mitogen-stimulated IgG2 production, confirming that PAF is essential for optimal responses. PAF-activated leukocytes produced gamma interferon (IFN-gamma), a Th1 cytokine that has been associated with IgG2 responses in previous studies. The monocyte-derived cytokines interleukin-12 (IL-12) and IL-18 are upstream of IFN-gamma production, and IgG2 production was suppressed by neutralizing antibodies against these proteins. In addition, PAF induced monocyte-derived dendritic cells (DC) but not macrophages (MPhi) to secrete IL-12 and IL-18. This observation was interesting because monocyte differentiation in LAgP subjects is skewed to the DC phenotype. Although other investigators have implicated IFN-gamma in IgG2 production, its precise role in this response is controversial. Our studies suggest that IFN-gamma induces isotype switching to IgG2 but only in concert with the Th2 cytokine IL-4. Thus, it appears that the unique PAF metabolism of LAgP monocytes or DC promotes Th1 responses that are essential for optimal IgG2 antibody production. As IgG2 antibodies opsonize oral bacteria and promote their clearance and destruction, these alterations in PAF metabolism may be essential for limiting disease severity in LAgP patients.

The immunosuppressant FTY720 is phosphorylated by sphingosine kinase type 2.

The potent immunosuppressive drug FTY720, a sphingosine analog, induces redistribution of lymphocytes from circulation to secondary lymphoid tissues. FTY720 is phosphorylated in vivo and functions as an agonist for four G-protein-coupled sphingosine-1-phosphate receptors. The identity of the kinase that phosphorylates FTY720 is still not known. Here we report that although both sphingosine kinase type 1 (SphK1) and type 2 (SphK2) can phosphorylate FTY720 with low efficiency, SphK2 is much more effective than SphK1. FTY720 inhibited phosphorylation of sphingosine catalyzed by SphK2 to a greater extent than it inhibits SphK1. Thus, SphK2 may be the relevant enzyme that is responsible for in vivo phosphorylation of FTY720.

Prostaglandin E2-mediated regulation of immunoglobulin G2 via interferon gamma.

BACKGROUND: Patients with localized aggressive periodontitis (LAgP) produce elevated levels of IgG2 both in vivo and in vitro. Responses to the periodontitis-associated pathogens Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis are dominated by IgG2, and these IgG2 responses are associated with reduced extent and severity of disease. Little is known about regulation of the IgG2 subclass, although prostaglandin E2 (PGE2) (a mediator known to polarize responses toward Th-2) and interferon (IFN)-gamma (a Th-1 mediator) both promote IgG2 production. This unusual relationship prompted the hypothesis that, in certain circumstances, PGE2 enhances rather than inhibits IFN-gamma production. METHODS: To test this hypothesis, indomethacin (IND)-treated peripheral blood mononuclear cell (PBL) cultures from healthy volunteers were stimulated with pokeweed mitogen (PWM), and the cultures were manipulated by adding PGE2, rIFN-gamma, rIL-Ialpha, rIL-1beta, rIL-6, or rIL-12. Production of IgG1, IgG2, IFN-gamma, and PGE2 was monitored by enzyme-linked immunosorbent assay. RESULTS: Indomethacin treatment inhibited IgG1 and IgG2 production, and PGE2 restored both immunoglobulins in PWM-stimulated cultures. Remarkably, addition of IFN-gamma also restored IND-suppressed IgG2 but not IgG1. In contrast, addition of rIL (interleukin)-1alpha, rIL-1beta, rIL-6, or rIL-12 did not restore IgG2 responses. Furthermore, IND suppressed IFN-gamma production and PGE2 increased IFN-gamma levels. Kinetic studies indicate that PGE2 production occurred on the first day of culture, followed by IFN-gamma two days later. CONCLUSIONS: These findings support the concept that in certain circumstances, PGE2 production can lead to increased IFN-gamma and that this IFN-gamma selectively promotes IgG2 responses. These data suggest that the elevated PGE2 observed in LAgP patients may contribute to increased IFN-gamma production and help explain elevated IgG2 responses.

Differential expression of platelet-activating factor acetylhydrolase in macrophages and monocyte-derived dendritic cells.

Although macrophages (Mphi) and monocyte-derived dendritic cells (MDDC) come from a common precursor, they are distinct cell types. This report compares the two cell types with respect to the metabolism of platelet-activating factor (PAF), a biologically active lipid mediator. These experiments were prompted by our studies of localized juvenile periodontitis, a disease associated with high IgG2 production and a propensity of monocytes to differentiate into MDDC. As the IgG2 Ab response is dependent on PAF, and MDDC selectively induce IgG2 production, we predicted that PAF levels would be higher in MDDC than in Mphi. To test this hypothesis, human MDDC were prepared by treating adherent monocytes with IL-4 and GM-CSF, and Mphi were produced by culture in M-CSF. Both Mphi and MDDC synthesized PAF; however, MDDC accumulated significantly more of this lipid. We considered the possibility that PAF accumulation in MDDC might result from reduced turnover due to lower levels of PAF acetylhydrolase (PAFAH), the enzyme that catabolizes PAF. Although PAFAH increased when monocytes differentiated into either cell type, MDDC contained significantly less PAFAH than did Mphi and secreted almost no PAFAH activity. The reduced levels of PAFAH in MDDC could be attributed to lower levels of expression of the enzyme in MDDC and allowed these cells to produce PGE(2) in response to exogenous PAF. In contrast, Mphi did not respond in this manner. Together, these data indicate that PAF metabolism may impinge on regulation of the immune response by regulating the accessory activity of MDDC.

Monocyte differentiation in localized juvenile periodontitis is skewed toward the dendritic cell phenotype.

Several lines of evidence indicate that the monocytes of subjects with localized juvenile periodontitis (LJP) are functionally distinct from cells of age- and race-matched nonperiodontitis (NP) subjects. Among the abnormalities are the propensity to secrete large amounts of prostaglandin E(2) and the induction of immunoglobulin G2 (IgG2) antibodies. The experiments described here were performed to further characterize the LJP monocytes and to determine if these cells mature differently than NP monocytes. When adherent monocytes from LJP subjects were cultured in the presence of human serum, both macrophages and cells with the morphology of immature monocyte-derived dendritic cells (MDDC) were observed. Within 4 days the prevalence of the immature MDDC was approximately twofold higher in LJP cultures than in NP cultures. In addition to their dendritic morphology, these cells were CD11c(+) and CD14(-) or CD14(low) and stimulated potent autologous mixed leukocyte reactions, consistent with differentiation to the MDDC phenotype. Like LJP monocytes, cultures of MDDC generated with interleukin-4 and granulocyte-macrophage colony-stimulating factor selectively induced IgG2 in cultures of pokeweed mitogen-stimulated NP leukocytes. Together, these data suggest that the monocytes of LJP subjects have a propensity to differentiate into MDDC and that this differentiation may be related to the high levels of IgG2 that are observed in the sera of LJP subjects. As high levels of circulating IgG2 are correlated with less severe disease, the propensity of LJP monocytes to differentiate into MDDC may have important implications for both the host response against oral pathogens and the progression of LJP.