Novel structural determinants of the human neutrophil chemotactic activity of leukotriene B.

A specific 5(S),12(R)-dihydroxy-eicosa-6,8,10(trans/trans/cis), 14(cis)-tetraenoic acid, designated leukotriene B, is generated by the lipoxygenation and subsequent enzymatic hydration of arachidonic acid in a variety of leukocytes. Leukotriene B elicits a maximal human neutrophil chemotactic response in vitro which is similar in magnitude to those evoked by the chemotactic fragment of the fifth component of complement, C5a, synthetic formyl-methionyl peptides, and 5-hydroxy-eicosatetraenoic acid (5-HETE). The neutrophil chemotactic potency of purified leukotriene B, assessed by the 50% effective concentration of 6 x 10(-9) M, is equivalent to that of C5a, but is up to 100-fold greater than that of 5-HETE and of other natural di-HETE isomers. 5(S),12(R)-di-hydroxy-eicosa-6,8,10(all-trans),14(cis)-tetraenoic acid, which differs from leukotriene B only in having a trans-double bond in place of a cis-double bond in the triene portion of the molecule, and acetyl-leukotriene B are significantly less potent neutrophil chemotactic factors than leukotriene B, which indicates that both the conjugated double bonds and the free hydroxyl-group(s) are functionally critical determinants. The capacity of acetyl-leukotriene B to inhibit competitively and selectively the human neutrophil chemotactic response to equimolar concentrations of leukotriene B suggests the existence of a specific subset of receptors for this potent lipid mediator

Generation of unique mono-hydroxy-eicosatetraenoic acids from arachidonic acid by human neutrophils.

Incubation of [3H]arachidonic acid with the 17,000-g supernatant from homogenates of human neutrophils in the presence of indomethacin generated the unique metabolites 9-OH-5,7,11,14-eicosatetraenoic acid (9-HETE) and 8-HETE, in addition to 12-HETE, 11-HETE and 5-HETE. The human neutrophil chemotactic activity of the HETE products exhibited a rank-order of potency with 5-HETE greater than 8-HETE = 9-HETE greater than 11-HETE = 12-HETE. The expression of chemokinetic activity as well as chemotactic activity suggested that the endogenous production of these principles may influence the mobility of human neutrophils

Stimulus specificity of the generation of leukotrienes by dog mastocytoma cells.

Isolated dog mastocytoma cells sensitized with dog anti-ragweed IgE and challenged with ragweed antigen or incubated with ionophore A23187 or the carboxy-terminal dodecapeptide of platelet factor 4, PF4(59-70), release histamine and concurrently generate leukotrienes B4, C4, and D4. In contrast, the exposure of mastocytoma cells to 0.1-3 micrograms/ml of 15-hydroxyeicosatetraenoic acid (15-HETE) stimulates selectively the generation of leukotrienes, in the absence of histamine release, while 0.1-1 micrograms/ml of compound 48/80 releases histamine without enhancing the generation of leukotrienes. That natural stimuli are capable of selectively activating one synthetic or secretory compartment of mast cells suggests that separate subsets of receptors as well as different biochemical events may serve to mobilize each class of mediators

Modulation of human lymphocyte function by C3a and C3a(70-77).

Human C3a and the synthetic octapeptide C3a (70-77), which retains the activities of an anaphylatoxin, inhibit in a concentration-dependent manner the generation of leukocyte inhibitory factor (LIF) activity by human mononuclear leukocytes and T lymphocytes cultured with the mitogens phytohemagglutinin (PHA) or concanavalin A (Con A) or the antigen streptokinase-streptodornase (SK-SD). The generation of LIF activity was inhibited by 50% by 10(-8) M C3a or C3a(70-77) with PHA or Con A as the stimulus, whereas a more than 10-fold higher concentration of C3a(70-77) than C3a was required to achieve the same level of suppression with SK-SD as the stimulus. Similar concentrations of C3a(70-77) inhibited to the same extent the migration of T lymphocytes stimulated by alpha-thioglycerol of Con A. Neither C3a nor C3a(70-77) altered significantly the uptake of [3H]thymidine by human mononuclear cells exposed to PHA, Con A, or SK-SD. The capacity of C3a(70-77)-Sepharose,m but not Sepharose alone, to adsorb or inactivate mononuclear leukocytes required for the generation of LIF activity established a direct interaction. Analysis of the lymphocytes in the effluent from C3a(70-77)-Sepharose columns, using monoclonal antibodies to surface antigens, showed a selective depletion of the helper/inducer population of lymphocytes. C3a might represent an important mediator of the functionally selective regulation of human T lymphocyte activities by the complement system

A neutrophil-dependent pathway for the generation of a neutral peptide mediator: partial characterization of components and control by alpha-1-antitrypsin.

A biologically active neutral peptide mediator is cleaved from a plasma protein substrate by an alpha-1-antitrypsin-inhibitable serine protease apparently residing on the membrane of the human neutrophil. The peptide mediator has an approximate mol wt of 1,000, and is distinguished from the kinin peptides by a neutral isoelectric point, susceptibility to inactivation by trypsin as well as chymotrypsin and activity on the isolated, atropinized, and antihistamine-treated guinea pig ileum with relatively little action on the estrous rat uterus. The neutrophil protease is fully inhibitable by DFP, trypsin inhibitors from lima or soy bean, and alpha-1-antitrypsin and is associated with the high mol wt fragments of the neutrophil and not the nuclear, lysosomal, or cytoplasmic subcellular fraction. The substrate has an approximate mol wt of 90,000 and is chromatographically separable from kininogen. The exquisite sensitivity of the neutrophil protease to alpha-1-antitrypsin was established both by inhibition with highly purified alpha-1-antitrypsin and by the inability of the protease to generate detectable neutral peptide in a homozygous (ZZ) alpha-1-antitrypsin-deficient patient without heat inactivation of the residual inhibitor. On the other hand, plasma from a (null) alpha-1-antitrypsin-deficient patient supported neutral peptide generation and revealed an additional factor which inactivated neutral peptide

An acidic fibroblast growth factor protein generated by alternate splicing acts like an antagonist.

Polymerase chain reaction amplification of cDNA for acidic fibroblast growth factor in several lines of cultured human cells revealed two forms of mRNA. The novel smaller mRNA lacks the entire second coding exon of the acidic fibroblast growth factor gene, whereas the previously identified mRNA consists of three coding exons. The truncated variant of acidic fibroblast growth factor (aFGF') is only 60 amino acids long with an apparent molecular mass of 6.7 kD on sodium dodecyl sulfate gels in contrast to 18 kD for the full-length acidic fibroblast growth factor. aFGF' elicits only minimal fibroblast proliferation and antagonizes the effects of acidic fibroblast growth factor when added exogenously to or when coexpressed with aFGF in BALB/c/3T3 fibroblasts. Thus, the truncated variant of acidic fibroblast growth factor may provide fibroblasts with a unique mechanism for endogenous regulation of their responses to acidic fibroblast growth factor

Over-expression of lysophosphatidic acid receptor-2 in human invasive ductal carcinoma

INTRODUCTION: Lysophosphatidic acid (LPA) is a bioactive phospholipid with diverse effects on various cells. It interacts with at least three G-protein-coupled transmembrane receptors, namely LPA1, LPA2 and LPA3, whose expression in various tumours has not been fully characterized. In the present study we characterized the expression profile of LPA receptors in human breast cancer tissue and assessed the possible roles of each receptor. METHODS: The relative expression levels of each receptor's mRNA against β-actin mRNA was examined in surgically resected invasive ductal carcinomas and normal gland tissue using real-time RT-PCR. LPA2 expression was also examined immunohistochemically using a rat anti-LPA2 monoclonal antibody. RESULTS: In 25 cases normal and cancer tissue contained LPA1 mRNA at similar levels, whereas the expression level of LPA2 mRNA was significantly increased in cancer tissue as compared with its normal counterpart (3479.0 ± 426.6 versus 1287.3 ± 466.8; P < 0.05). LPA3 was weakly expressed in both cancer and normal gland tissue. In 48 (57%) out of 84 cases, enhanced expression of LPA2 protein was confirmed in carcinoma cells as compared with normal mammary epithelium by immunohistochemistry. Over-expression of LPA2 was detected in 17 (45%) out of 38 premenopausal women, as compared with 31 (67%) out of 46 postmenopausal women, and the difference was statistically significant (P < 0.05). CONCLUSION: These findings suggest that upregulation of LPA2 may play a role in carcinogenesis, particularly in postmenopausal breast cancer

Structural identification of oxidized acyl-phosphatidylcholines that induce platelet activation

Oxidation of low-density lipoprotein (LDL) generates proinflammatory and prothrombotic mediators that may play a crucial role in cardiovascular and inflammatory diseases. In order to study platelet-activating components of oxidized LDL 1-stearoyl-2-arachidonoyl-sn-glycero-3- phosphocholine, a representative of the major phospholipid species in LDL, the 1-acyl-phosphatidylcholines (PC), was oxidized by CuCl2 and H2O2. After separation by high-performance liquid chromatography, three compounds were detected which induced platelet shape change at low micromolar concentrations. Platelet activation by these compounds was distinct from the pathways stimulated by platelet-activating factor, lysophosphatidic acid, lyso-PC and thromboxane A(2), as evidenced by the use of specific receptor antagonists. Further analyses of the oxidized phospholipids by electrospray ionization mass spectrometry structurally identified them as 1-stearoyl-2-azelaoyl-sn-glycero-3-phosphocholine (m/z 694; SAzPC), 1-stearoyl-2-glutaroyl-snglycero-3- phosphocholine (m/z 638; SGPC), and 1-stearoyl-2-( 5-oxovaleroyl)-sn-glycero-3-phosphocholine (m/z 622; SOVPC). These observations demonstrate that novel 1-acyl-PC which had previously been found to stimulate interaction of monocytes with endothelial cells also induce platelet activation, a central step in acute thrombogenic and atherogenic processes. Copyright (C) 2005 S. Karger AG, Basel

Lysophosphatidic acid-3 receptor-mediated feed-forward production of lysophosphatidic acid: an initiator of nerve injury-induced neuropathic pain

<p>Abstract</p> <p>Background</p> <p>We previously reported that intrathecal injection of lysophosphatidylcholine (LPC) induced neuropathic pain through activation of the lysophosphatidic acid (LPA)-1 receptor, possibly via conversion to LPA by autotaxin (ATX).</p> <p>Results</p> <p>We examined <it>in vivo </it>LPA-induced LPA production using a biological titration assay with B103 cells expressing LPA₁receptors. Intrathecal administration of LPC caused time-related production of LPA in the spinal dorsal horn and dorsal roots, but not in the dorsal root ganglion, spinal nerve or sciatic nerve. LPC-induced LPA production was markedly diminished in ATX heterozygotes, and was abolished in mice that were deficient in LPA₃, but not LPA₁or LPA₂receptors. Similar time-related and LPA₃receptor-mediated production of LPA was observed following intrathecal administration of LPA. In an <it>in vitro </it>study using spinal cord slices, LPA-induced LPA production was also mediated by ATX and the LPA₃receptor. Intrathecal administration of LPA, in contrast, induced neuropathic pain, which was abolished in mice deficient in LPA₁or LPA₃receptors.</p> <p>Conclusion</p> <p>These findings suggest that feed-forward LPA production is involved in LPA-induced neuropathic pain.</p