Leslie R. Ballou

The febrile response to lipopolysaccharide is blocked in cyclooxygenase-2-/-, but not in cyclooxygenase-1-/- mice

Various lines of evidence have implicated inducible cyclooxygenase-2 (COX-2) in fever production. Thus, its expression is selectively enhanced in brain after peripheral exogenous (e.g., lipopolysaccharide [LPS]) or endogenous (e.g., interleukin-1) pyrogen administration, while selective COX-2 inhibitors suppress the fever induced by these pyrogens. In this study, we assessed the febrile response to LPS of congenitally constitutive COX-1 (COX-1-/-) and COX-2 (COX-2-/-)-deficient C57BL/6J-derived mice. COX-1+/- and COX-2+/- mice were also evaluated; controls were wild-type C57BL/6J mice (Jackson Labs.). All the animals were pretrained daily for two weeks to the experimental procedures. LPS was injected intraperitoneally at 1 microgram/mouse; pyrogen-free saline (PFS) was the vehicle and control solution. Core temperatures (Tcs) were recorded using thermocouples inserted 2 cm into the colon. The presence of the COX isoforms was determined in cerebral blood vessels immunocytochemically after the experiments, without knowledge of the functional results. The data showed that the wild-type, COX-1+/-, and COX-1-/- mice all responded to LPS with a 1 degrees C rise in Tc within 1 h; the fever gradually abated over the next 4 h. By contrast, COX-2+/- and COX-2-/- mice displayed no Tc rise after LPS. PFS did not affect the Tc of any animal. It would appear therefore that COX-2 is necessary for LPS-induced fever production.

Ceramide signalling and the immune response

Ceramide, produced through either the induction of SM hydrolysis or synthesized de novo transduces signals mediating differentiation, growth, growth arrest, apoptosis, cytokine biosynthesis and secretion, and a variety of other cellular functions. A generalized ceramide signal transduction scheme is shown in Fig. 2 in which ceramide is generated through the activation of distinct SMases residing in separate subcellular compartments in response to specific stimuli. Clearly, specificity of cellular responses to ceramide depends upon many factors which include the nature of the stimulus, co-stimulatory signals and the cell type involved. Ceramide derived from neutral SMase activation is thought to be involved in modulating CAPK and MAP kinases, PLA2 (arachidonic acid mobilization), and CAPP while ceramide generated through acid SMase activation appears to be primarily involved in NF-kappa B activation. While there is no apparent cross-talk between these two ceramide-mediated signalling pathways, there is likely to be significant cross-talk between ceramide signalling and other signal transduction pathways (e.g., the PKC and MAP kinase pathways). Other downstream targets for ceramide action include Cox, IL-6 and IL-2 gene expression, PKC zeta, Vav, Rb, c-Myc, c-Fos, c-Jun and other transcriptional regulators. Many, if not all, of these ceramide-mediated signalling events have been identified in the various cells comprising the immune system and are integral to the optimal functioning of the immune system. Although the role of the SM pathway and the generation of ceramide in T and B lymphocytes have only recently been recognized, it is clear from these studies that signal transduction through SM and ceramide can strongly affect the immune response, either directly through cell signalling events, or indirectly through cytokines produced by other cells as the result of signalling through the SM pathway. An overview of the signalling mechanisms coupling ceramide to the modulation of the immune response is depicted in Fig. 3 and shows how ceramide may play pivotal roles in regulating a number of complex processes. The SM pathway represents a potentially valuable focal point for therapeutic control of immune responses, perhaps for either enhancement of the activity of T cells in the elimination of tumors, or the down-regulation of lymphocyte function in instances of autoimmune disease. The recent explosion of knowledge regarding ceramide signalling notwithstanding, a number of critical questions need to be answered before a comprehensive, mechanistic understanding can be formulated relative to the incredibly varied effects of ceramide on cell function. For example, (i) how is a structurally simple molecule like ceramide able to mediate so many different, and sometimes paradoxical, physiological responses ranging from cell proliferation and differentiation to inhibition of cell growth and apoptosis, (ii) what are the molecular identities and modes of activation of the various SMase isoforms, (iii) what determines the distribution of the unique isoforms of SMase in cells of different lineages or at different stages of differentiation, (iv) what is the relative contribution of ceramide generated through SM hydrolysis versus de novo synthesis, and (v) by what means does ceramide interact with specific intracellular targets? Although a number of ceramide-activatable kinases, phosphatases, and their protein substrates have been identified, a more extensive search for additional cellular targets will be indispensable in determining the phosphorylation cascades linking the activation of the SM pathway to the regulation of nuclear events. Clearly, cross-talk between ceramide-induced signal transduction cascades and other signalling pathways adds to the inherent difficulty in distinguishing the specific effects of complex, intertwining signalling pathways.

p53-mediated induction of Cox-2 counteracts p53- or genotoxic stress-induced apoptosis

The identification of transcriptional targets of the tumor suppressor p53 is crucial in understanding mechanisms by which it affects cellular outcomes. Through expression array analysis, we identified cyclooxygenase 2 (Cox‐2), whose expression was inducible by wild‐type p53 and DNA damage. We also found that p53‐induced Cox‐2 expression results from p53‐mediated activation of the Ras/Raf/MAPK cascade, as demonstrated by suppression of Cox‐2 induction in response to p53 by dominant‐negative Ras or Raf1 mutants. Furthermore, heparin‐binding epidermal growth factor‐like growth factor (HB‐ EGF), a p53 downstream target gene, induced Cox‐2 expression, implying that Cox‐2 is an ultimate effector in the p53→HB‐EGF→Ras/Raf/MAPK→Cox‐2 pathway. p53‐induced apoptosis was enhanced greatly in Cox‐2 knock‐out cells as compared with wild‐type cells, suggesting that Cox‐2 has an abrogating effect on p53‐induced apoptosis. Also, a selective Cox‐2 inhibitor, NS‐398, significantly enhanced genotoxic stress‐induced apoptosis in several types of p53+/+ normal human cells, through a caspase‐dependent pathway. Together, these results demonstrate that Cox‐2 is induced by p53‐mediated activation of the Ras/Raf/ERK cascade, counteracting p53‐mediated apoptosis. This anti‐apoptosis effect may be a mechanism to abate cellular stresses associated with p53 induction.

Obesity is induced in mice heterozygous for cyclooxygenase-2

In mice heterozygous for the cyclooxygenase-2 gene (COX-2+/-) the body weight was enhanced by 33% as compared to homozygous COX-2-/- mice. The weights of the gonadal fat pads in COX-2+/- mice were enhanced by 3.5 to 4.7 fold as compared to COX-2-/- mice and by 1.5 to 3.5 fold as compared to wild-type controls+/+ Serum leptin levels and leptin release by cultured adipose tissue of COX-2+/- mice were both elevated as compared to either control or COX-2-/- animals. The basal release of PGE2 or 6 keto PGF1alpha per fat pad over a 24 h incubation of adipose tissue was reduced by 80% and 95% respectively in tissue from COX-2-/- mice. NS-398, a specific COX-2 inhibitor, inhibited leptin release by 27% in adipose tissue from control mice, 31% in tissue from COX-1-/- mice and by 23% in tissue from COX-2+/- mice while having no effect on leptin release by adipose tissue from COX-2-/- mice. These data indicate that heterozygous COX-2 mice develop obesity which is not secondary to a defect in leptin release by adipose tissue.

Sphingolipids and cell function

Abstract In this short article, Leslie Ballou draws together the various strands of evidence that suggest that sphingolipids function as a unique class of second messenger molecules, capable of modulating a wide variety of cellular processes.

Intracerebroventricular interleukin-6, macrophage inflammatory protein-1β and IL-18: pyrogenic and PGE2-mediated?

This study was undertaken to determine whether cyclooxygenase (COX)-2, the critical enzyme in the production of febrigenic prostaglandin (PG) E(2), may be involved centrally in the fever induced in mice by homologous interleukin (IL)-6, macrophage inflammatory protein (MIP)-1 beta, and interleukin (IL)-18, a member of the pyrogenic IL-1 beta family. To this end, the core temperatures (Tc) of COX-1 and COX-2 gene-ablated mice and of their normal wild-type (WT) counterparts were recorded after intracerebroventricular (i.c.v.) challenge with recombinant murine (rm) IL-6 (10 ng/mouse), rmMIP-1 beta (20 pg/mouse), rmIL-18 (0.01-1 microgram/mouse), rmIL-1 beta (positive control; 0.1 microgram/mouse), or their vehicle (0.1% bovine serum albumin [BSA] in sterile phosphate-buffered saline [PBS]; 5 microl/mouse). rmIL-6 caused a approximately 1 degrees C T(c) rise in WT mice that peaked at approximately 120 min and gradually recovered over the next 3 h; COX-1(-/-) mice exhibited a relatively faster (peak at 45 min) and shorter (recovery at 150 min) febrile course, whereas COX-2(-/-) mice did not develop fever. rmMIP-1 beta induced a 1 degrees C fever (peak at 60 min) with a long time course (recovery incomplete at 300 min) in both WT and COX-2(-/-) mice; COX-1(-/-) mice displayed a quick-onset (peak at 40 min) and shorter (recovery at approximately 240 min) fever. rmIL-18 did not cause any thermal response at any dose whether administered intraperitoneally (i.p.) or i.c.v. in WT mice; COX gene-ablated mice, therefore, were not tested. These data indicate that COX-2-dependent PGE(2) is critical for the febrile response to IL-6, but not to MIP-1 beta. IL-18 i.p. or i.c.v. is not pyrogenic.

The tissue-specific, compensatory expression of cyclooxygenase-1 and -2 in transgenic mice

Prostaglandins are essential regulators of tissue homeostasis, reproduction and inflammation. We have recently shown that cells derived from cyclooxygenase (COX)-deficient mice express higher, compensatory levels of the remaining COX isozyme [Kirtikara et al., J. Exp. Med., 187, 517 (1998)]. To assess this compensatory expression phenomenon in vivo, we quantified COX-1 and COX-2 mRNA levels in various organs of COX-1- and COX-2-ablated mice using a reverse transcriptase-polymerase chain reaction (RT-PCR) method. We found that COX-1 and COX-2 mRNAs in the brains of COX-ablated mice were elevated > 2-fold compared with wild-type (WT) animals. COX-2 mRNA was enhanced approximately 2-fold in the kidneys and stomachs of COX-1-deficient mice while COX-1 expression remained unchanged. Conversely, the livers of COX-2-deficient mice expressed 15-fold higher COX-1 mRNA levels, while hepatic COX-2 mRNA levels were not significantly altered in the COX-1-ablated mice. Steady state levels of COX-1 and COX-2 mRNAs in the hearts, lungs and spleens of WT, COX-1- and COX-2-deficient mice were indistinguishable from each other. Peritoneal macrophages isolated from COX-1- and COX-2-ablated mice also expressed significantly higher steady-state levels of cytoplasmic phospholipase A2 and 5-lipooxygenase mRNAs suggesting a global upregulation of eicosanoid biosynthetic pathways in COX-deficient mice. These data suggest that expression of both COX-1 and COX-2 can be re-programmed to compensate for the lack of both alleles of the alternate COX gene in transgenic mice.

Cyclooxygenase‐1 and cyclooxygenase‐2 in the mouse ductus arteriosus: individual activity and functional coupling with nitric oxide synthase

Prenatal patency of the ductus arteriosus is maintained by prostaglandin (PG) E2, conceivably in concert with nitric oxide (NO). Local PGE2 formation is sustained by cyclooxygenase‐1 (COX1) and cyclooxygenase‐2 (COX2), a possible exception being the mouse in which COX1, or both COXs, are reportedly absent. Here, we have examined the occurrence of functional COX isoforms in the near‐term mouse ductus and the possibility of COX deletion causing NO upregulation. COX1 and COX2 were detected in smooth muscle cells by immunogold electronmicroscopy, both being located primarily in the perinuclear region. Cytosolic and microsomal PGE synthases (cPGES and mPGES) were also found, but they occurred diffusely across the cytosol. COX1 and, far more frequently, COX2 were colocalised with mPGES, while neither COX appeared to be colocalized with cPGES. The isolated ductus from wild‐type and COX1−/− mice contracted promptly to indomethacin (2.8 μM). Conversely, the contraction of COX2−/− ductus to the same inhibitor started only after a delay and was slower. NG‐nitro‐L‐arginine methyl ester (L‐NAME, 100 μM) weakly contracted the isolated wild‐type ductus. Its effect, however, increased three‐ to four‐fold after deleting either COX, hence equalling that of indomethacin. In vivo, the ductus was patent in all mice foetuses, whether wild‐type or COX‐deleted. Likewise, no genotype‐related difference was noted in its postnatal closure. We conclude that the mouse ductus has a complete system for PGE2 synthesis comprising both COX1 and COX2. The two enzymes respond differently to indomethacin but, nevertheless, deletion of either one results in NO upregulation. PGE2 and NO can function synergistically in keeping the ductus patent.

Both Constitutive and Inducible Prostaglandin H Synthase Affect Dermal Wound Healing in Mice

In an attempt to define the roles of prostaglandin H synthase 1 (PGHS-1, cyclooxygenase-1, COX-1) and prostaglandin H synthase 2 (PGHS-2, cyclooxygenase-2, COX-2) in wound healing, we investigated the healing of incisional dermal wounds in wild-type, PGHS-1 null, and PGHS-2 null mice. We measured tensile strength of the wounds, levels of PGHS-1 and PGHS-2 mRNA in the wound site, and histologic markers for the inflammatory, proliferative, and remodeling phases of wound healing. Although no gross visible differences were noted among healed wounds of the different mouse types, measurement of tensile strength showed that both PGHS-1 and PGHS-2 null wounds were weaker (75% and 70%, respectively) than wild-type wounds at 12 days after incision. At Day 8 the endothelial staining was 70% greater in the wounds of PGHS-2 null mice compared with their wild-type counterparts. In contrast at Day 12, staining for macrophages and myofibroblasts was less in PGHS-1 null wounds compared with wild-type and PGHS-2 null tissue. Compensatory expression of the alternate PGHS mRNA could be demonstrated by RT-PCR in the wounds of PGHS null mice on Days 1 and 4. We conclude that both PGHS-1 and PGHS-2 genes play distinct roles in the process of dermal wound healing.

An accessory role for ceramide in interleukin-1beta induced prostaglandin synthesis.

Interleukin-1β (IL-1) is a potent inducer of prostaglandin E2 (PGE2) synthesis. We previously showed that ceramide accumulates in fibroblasts treated with IL-1 and that it enhances IL-1-induced PGE2 production. The present study was undertaken to determine the mechanism(s) by which ceramide and IL-1 interact to enhance PGE2 production by examining their respective effects on the rate-limiting enzymes in PGE2 synthesis, cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and cytosolic phospholipase A2 (cPLA2). IL-1-induced PGE2 synthesis required ω8 h even though COX-1 was constitutively expressed (both mRNA and protein) and enzymatically active in untreated cells. Conversely, COX-2 mRNA was barely detectable in untreated cells but within 2 h, ceramide or IL-1 alone induced a 5 and 20 fold increase in COX-2 mRNA, respectively. However, IL-1 induced COX-2 protein synthesis was only detectable 6-7 h after maximal COX-2 mRNA induction; COX-2 protein accumulation was not induced by ceramide alone. Ceramide however, reduced the length of time required for IL- 1 to induce COX-2 protein accumulation and increased COX-2 protein accumulation. IL-1 induced a 15 fold increase in COX-1 mRNA including an alternatively spliced form of COX-1. IL-1, but not ceramide induced cPLA2 mRNA and protein expression which corresponded with the initiation of PGE2 synthesis. These observations indicate that, (1) while either ceramide or IL-1 rapidly induced COX-2 mRNA, COX-2 protein only accumulated in IL- 1 treated cells after a delay of 6-7 h, (2) IL-1-induced PGE2 synthesis required both COX-2 and cPLA2 protein synthesis and, (3) ceramide enhanced (temporally and quantitatively) IL-1-induced COX-2 protein accumulation resulting in enhanced PGE2 production.