12/15-Lipoxygenase (LOX) mediates immune-regulatory activities not accounted for by its known free acid eicosanoids, suggesting that additional lipids may be generated by activated cells. To characterize novel LOX-derived lipids, a lipidomic approach was utilized. Ionophore-activated interleukin-4-treated human peripheral monocytes generated up to 10-fold more esterified 15-hydroxyeicosatetraenoic acid (15-HETE) than free in a phosphatidylinositol 3-kinase-and protein kinase C-sensitive manner. Precursor scanning electrospray ionization/tandem spectroscopy for m/z 319 (HETE, [M-H] ؊ ) showed 4 ions at m/z 738, 764, 766, and 782 that were identified using tandem spectroscopy and MS3 as specific diacyl and plasmalogen 15-HETE phosphatidylethanolamines. Using H 2 18 O water, the compounds were shown to form by direct oxidation of endogenous phosphatidylethanolamine (PE) by 15-LOX, with PE being the preferred phospholipid pool containing 15-HETE. Similarly, human platelets generated 4 analogous PE lipids that contained 12-HETE and increased significantly in response to ionophore, collagen, or convulxin. These products were retained in the cells, in contrast to free acids, which are primarily secreted. Precursor scanning of platelet extracts for the major platelet-derived prostanoid, thromboxane B2 (m/z 369.2), did not reveal PE esters, indicating that this modification is restricted to the LOX pathway. In summary, we show formation of PE-esterified HETEs in immune cells that may contribute to LOX signaling in inflammation.

Summary Metalloproteinases (MMPs) participate in extracellular matrix remodelling and regulatory signalling during chronic inflammatory states such as atherosclerosis formation. However, the sources and mediators of MMP upregulation need clarification. We investigated whether proinflammatory mouse T helper type 1 (Th1) lymphocytes are more active in MMP secretion than naïve Th0 or anti‐inflammatory Th2 phenotypes, in the absence of specific antigenic stimulation, under baseline conditions and after contact with irradiated macrophages. We also compared the effect of Th0, Th1 or Th2 lymphocyte‐conditioned medium and irradiated lymphocytes on MMP production from macrophages. Finally, we investigated whether CD40–CD40 ligand (CD40L) interactions were involved in T‐cell‐stimulated MMP secretion from macrophages. Under baseline conditions, MMP‐2 messenger RNA (mRNA) and protein levels were greater in Th1 than Th0 or Th2 lymphocytes; MMP‐9 mRNA, but not protein, was also upregulated. In the presence of irradiated macrophages MMP‐2 and MMP‐9 production from Th1 and Th2 was greater than from Th0 lymphocytes. Conditioned media from Th1 but not Th0 or Th2 cells increased MMP‐9 secretion from macrophages. Irradiated Th1 lymphocytes stimulated both MMP‐2 and MMP‐9 secretion from macrophages more than irradiated Th2 or Th0 cells; this activation was independent of CD40–CD40L interaction. These findings demonstrate for the first time greater MMP secretion by Th1 than Th2 or Th0 lymphocytes and their greater ability to upregulate macrophage MMP secretion in the absence of specific antigenic stimulation. These mechanisms could promote matrix turnover in inflammatory states and, for example, promote atherosclerotic plaque rupture.

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12/15-Lipoxygenase (12/15-LOX) plays a pathogenic role in atherosclerosis. To characterize whether 12/15-LOX also contributes to endothelial dysfunction and hypertension, regulation of vessel tone and angiotensin II (ang II) responses were characterized in mice deficient in 12/15-LOX. There was a twofold increase in the magnitude of l-nitroarginine-methyl ester-inhibitable, acetylcholine-dependent relaxation or phenylephrine-dependent constriction in aortic rings isolated from 12/15-LOX(-/-) mice. Plasma NO metabolites and aortic endothelial NO synthase (eNOS) expression were also elevated twofold. Angiotensin II failed to vasoconstrict 12/15-LOX(-/-) aortic rings in the absence of L-nitroarginine-methyl ester, and ang II impaired acetylcholine-induced relaxation in wild-type, but not 12/15-LOX(-/-) rings. In vivo, 12/15-LOX(-/-) mice had similar basal systolic blood pressure measurements to wild type, however, blood pressure elevations in response to ang II infusion (1.1 mg/kg/day) were significantly attenuated (maximal pressure, 143.4 +/- 4 mmHg versus 122.1 +/- 5.3 mmHg for wild type and 12/15-LOX(-/-), respectively). In contrast, vascular hypertrophic responses to ang II, and ang II type 1 receptor (AT1-R) expression were similar in both strains. This study shows that 12/15-LOX(-/-) mice have increased NO biosynthesis and impaired ang II-dependent vascular responses in vitro and in vivo, suggesting that 12/15-LOX signaling contributes to impaired NO bioactivity in vascular disease in vivo.

Gap junction channels constructed of connexins (Cxs) are expressed by peripheral and secondary lymphoid organ-derived lymphocytes. These channels in the plasma membrane play key roles in a range of lymphocyte functions exemplified by the synthesis and secretion of Igs and cytokines and during transmigration across the endothelium. Most recently, their involvement in antigen crosspresentation has also been established. We report here for the first time the expression of mRNA and protein encoding Cx43 in mouse-derived CD4 + Th0, Th1, and Th2 lymphocyte subpopulations and demonstrate the establishment gap junction channel formation with primary macrophages in vitro. We show that this mode of direct communication is particularly favored in Th1-macrophage interactions and that LPS inhibits lymphocyte-macrophage cross-talk independently of the subset of lymphocyte involved. Our work suggests that gap junctionmediated communication can be modulated in the absence of specific antigenic stimulation. Therefore, a further mechanism featuring gap junction-mediated communication may be implicated in immune regulation. Keywords inflammation; intercellular; flow cytometry; lymphocytes Intercellular communication underpins cellular activation and various functions in mammals. In the hematopoietic and immune systems, direct cell-to-cell interactions influence cell phenotypic and functional characteristics such as those involved in blood formation or antigen-specific immune responses. These processes involve subsets of interacting cells, the surrounding signaling environment, and the functional outcomes of receptor-ligand interactions. One of the key channels underpinning intercellular communication are gap junctions, which are the focus of research by various groups who have shown their participation in leukocyte biology and the generation of immune responses [1][2][3][4]. Despite increasing evidence for the role of Cx proteins and gap junction channels in inflammatory and immunological reactions, their expression and specific functional roles remain to be studied in lymphocyte subsets with distinct functional properties. Here, we describe for the first time the differential expression of mRNA and protein encoding Cx43 in mousederived CD4 + Th lymphocyte subpopulations in vitro. We show that all of these cells can communicate with macrophages via gap junctions and that such cross-talk is particularly favored in Th1-macrophage interactions.A well-established methodology was used to obtain differentiated CD4 + Th1 and Th2 lymphocyte subpopulations in the absence of antigenic stimulation using Th0 (naïve) lymphocytes isolated from the spleen of specific pathogen-free (SPF) CD-1 male mice [13]. Naïve CD4 + T cells were obtained by negative selection from the spleens of 8-to 10-weekold SPF male CD-1 mice (Harlam, UK). B cells, NK cells, monocytes, and CD8 + T cells were removed by incubating splenocytes with rat anti-mouse CD19, CD11c, and CD8 mAb (Serotec, UK) and anti-CD16/32 (PharMingen, San Diego, CA, USA) before incubati...