Altered leukotriene B4 levels by HL-60 cells after monocytic/macrophage differentiation

The differentiation of HL-60 human promyelocytic leukaemia cells into specific monocytic or granulocytic lineage cells depending of the inductor agent is accompanied by selective regulation of several key enzymes involved in the synthesis of eicosanoids. In this communication we have investigated the changes in arachidonic acid metabolic profiles during phorbol 12-myristate 13-acetate (PMA)-induced monocytic differentiation of HL-60 cells. Our results show that HL-60 cells have spontaneous capacity to synthesize large amounts of LTB₄, but PMA-differentiated cells lose the ability to release LTB₄. Significant differences are found between HL-60 cells and PMA-treated cells in basal conditions and under ionophore stimulation. The addition of LTB₄ at the time of PMA differentiation did not have effects on cell proliferation, but nordihydroguaiaretic acid (NDGA), a potent 5-lipoxygenase inhibitor, also inhibited HL-60 cell proliferation and did not have any effect on PMA-differentiated cell proliferation.     

Leukotriene B4 induces interleukin 5 generation from human T lymphocytes

Leukotriene B₄ (LTB₄) has been shown to affect several interleukin (IL)-linked functions of human lymphocytes. In this study, we investigated whether LTB₄ regulates IL-5 generation from human T cells and subsequently modulates eosinophil functions. Preincubation of T cells with very low concentrations (10^?12 to 10^?8 M ) of LTB₄ induced concentration-dependent IL-5 production, the event occurring after the first 24 h of cultivation. However, direct action of LTB₄ to IL-5 generation is strictly dependent on a preincubation with appropriate concentration of LTB₄. In contrast, the stereoisomer of LTB₄,5S,12S-dihydroxy-6,8,10,14-eicosatetraenoic acid showed no enhancement of IL-5 production. IL-5 released from LTB₄-primed T cells elicited sustained viability of mature eosinophils and reduced the content of eosinophil cationic protein in their crystalloid matrix by degranulation. These data suggest that LTB₄ induces bioactive IL-5 production from T cells and that the released IL-5 modulates eosinophil functions which might play a crucial role in eosinophil-linked allergic inflammatory process.     

Calcium ionophore and chemotactic peptide stimulation of peptidoleukotriene synthesis in DMSO-differentiated HL60 cells

The human promyelocytic leukemia cell line HL60 can be differentiated to mature granulocytes upon exposure to DMSO (1.3%, 6 days). The ability of these cells to metabolize arachidonic acid via the 5-lipoxygenase pathway to form 5-HETE, LTB₄, and 5,12-diHETEs, has been previously documented. However, the production of peptidoleukotrienes by DMSO-differentiated HL60 cells has not been previously reported. Arachidonic acid metabolites produced via 5-lipoxygenase were identified by reverse-phase, high-performance liquid chromatography, immunoreactivity specific for peptidoleukotriene, glutamyl transpeptidase transformation, characteristic UV spectra, and GC mass spectra. Leukotriene synthesis in the DMSO-differentiated HL60 cell is maximal at 5 min when stimulated with the calcium ioniphore, A23187 (1M), in the presence of calcium. These cells produce 12.94±1.8 ng/10⁶ cells of LTC₄ and 3.8±0.4 ng/10⁶ cells of LTB₄. LTC₄ and LTB₄ are also synthesized in the undifferentiated cell when stimulated with 1M A23187 and 1 mM Ca²⁺, but in much smaller quantities, i.e., 1.91±0.42 ng/10⁶ cells of LTC₄ and 0.41 ng±0.06/10⁶ cells of LTB₄. The synthetic chemotactic peptide, f-Met-Leu-Phe, also elicits formation of LTC₄ and LTB₄ in a dose-dependent manner in the presence of exogenously added calcium. Maximal stimulation of DMSO-differentiated cells with f-Met-Leu-Phe produces 2.5±0.2 ng of LTC₄ and 1.45±0.2 ng of LTB₄ per 10⁶ cells. The observation that DMSO-differentiated HL60 cells produce LTC₄, as well as other 5-lipoxygenase products, increases the utility of this cell line for unraveling the regulation of leukotriene biosynthesis by granulocytes.     

Inhibition of leukotriene B4(LTB4) by recombinant interleukin-1 receptor antagonist (IL-1RA) on human monocytes

Macrophages are a primary source of interleukin-1 (IL-1), a glycoprotein which plays an important and essential role in the immune response and inflammation. Cytokines stimulate many different cells to produce increasing amounts of arachidonic acid metabolites such as prostaglandins and leukotrienes. Recently, interleukin-1 receptor antagonist (IL-1ra), a natural inhibitor of IL-1 released by macrophages, has been reported to inhibit PGE₂. In accordance with these data our results show that the pretreatment, for 60 min, of purified human peripheral monocytes with IL-1ra at different concentrations (0.25–250 ng/ml) inhibits, in a dose-dependent manner, the generation of LTB₄ released after 10 min treatment with calcium ionophore A23187 (5 μM). The inhibition of LTB₄ synthesis by hrIL-1ra suggests the possibility that this new glycoprotein plays a modulatory role in immunity and inflammation.

     

Dual inhibition of 5-lipoxygenase/cyclooxygenase by a reconstituted homeopathic remedy; possible explanation for clinical efficacy and favourable gastrointestinal tolerability

Objective: In order to elucidate potential anti-inflammatory activities of Zeel comp. N and its constituents, the inhibition of the synthesis of Leukotriene B₄ (LTB₄) and Prostaglandin (PGE₂) by 5-lipoxygenase (5-LOX) and cyclo-oxygenase 1 and 2 (COX 1 and 2) respectively were examined in vitro.Materials: Human HL-60 cells, differentiated for 6–8 days with DMSO (1.2% v/v) were used for the 5-LOX assay. The COX activity assays were carried out with purified enzymes, COX 1 (ram seminal vesicles), COX 2 (sheep placenta) and with human THP-1 cells, differentiated for 24 h with PMA (50 nM).Methods: LTB₄ and PGE₂ production in the 5-LOX and COX assays respectively were determined by enzyme linked immunoassays.Results: A reconstituted Zeel comp. N combination as well as its constituent mother tinctures of Arnica montana, Sanguinaria canadensis and Rhus toxicodendron (Toxicodendron quercifolium) showed distinct inhibitory effects on the production of LTB₄ by 5-LOX (IC₅₀ values of 10, 20, 2 and 5 µg/ml respectively) and on the synthesis of PGE₂ by COX 1 (IC₅₀ values of 50, 80, 40 and 20 µg/ml respectively) and COX 2 enzymes (IC₅₀ values of 60, 110, 50 and 20 µg/ml respectively). The mother tincture of Solanum dulcamara inhibited the production of PGE₂ by COX 1 (IC₅₀ 40 µg/ml) and COX 2 (IC₅₀ 150 µg/ml) but not production of leukotriene LTB₄ by 5-LOX.Conclusions: The observed dual inhibition of both LOX- and COX-metabolic pathways may offer an explanation for the reported clinical efficacy and the favorable gastrointestinal tolerability of the original remedy Zeel comp. N.Received 10 April 2003; returned for revision 27 May 2003; accepted by W. B. van den Berg 24 November 2003     

Activation of human neutrophil LFA-1 (CD11a) by leukotriene B4

Homotypic aggregation (HA) of human neutrophils by the potent leukotactic factor, leukotriene B₄ (LTB₄), and phorbol myristate acetate (PMA) was evaluated by recording the net decrease in absorbency at 650 nm of suspensions of 10⁷ neutrophils/ml in a microtiter plate reader, which was found to correlate with microscopic evidence of aggregation. LTB₄-elicited HA was increased maximally by approximately one third above HA in buffer at 30 min, whereas PMA-induced HA reached a maximal level more than 21–fold higher than buffer control at 60 min. The involvement of LFA-1 in LTB₄-induced HA of neutrophils was suggested initially by the inhibitory effect of monoclonal anti-CD 18 and anti-CD11a antibodies. The binding to neutrophils of a monoclonal anti-LFA-1 antibody (NK1-L16) specific for an activation epitope of CD11a was increased a maximum of 28-fold and sixfold, respectively, after 1 and 5 min of preincubation with 10 nM LTB₄and fivefold after 5 min with PMA. Thus, both LTB₄ and PMA induce an activating conformational change in the CD11a adherence receptor of human neutrophils.     

Inhibition of leukotriene B4(LTB4) by recombinant interleukin-1 receptor antagonist (IL-1RA) on human monocytes

Macrophages are a primary source of interleukin-1 (IL-1), a glycoprotein which plays an important and essential role in the immune response and inflammation. Cytokines stimulate many different cells to produce increasing amounts of arachidonic acid metabolites such as prostaglandins and leukotrienes. Recently, interleukin-1 receptor antagonist (IL-1ra), a natural inhibitor of IL-1 released by macrophages, has been reported to inhibit PGE₂. In accordance with these data our results show that the pretreatment, for 60 min, of purified human peripheral monocytes with IL-1ra at different concentrations (0.25–250 ng/ml) inhibits, in a dose-dependent manner, the generation of LTB₄ released after 10 min treatment with calcium ionophore A23187 (5 M). The inhibition of LTB₄ synthesis by hrIL-1ra suggests the possibility that this new glycoprotein plays a modulatory role in immunity and inflammation.     

Leukotriene induction of TxB2 in cultured bovine aortic endothelial cells

The leukotrienes (LT) are potent mediators of inflammation, capable of inducing plasma leakage from postcapillary venules and vasoconstriction of terminal arterioles. Some microvascular effects may be attributable to LT stimulation of thromboxane (Tx) synthesis. Incubation of primary cultures of bovine aortic endothelial cells with buffer, LTB₄ (10^–8 M) or LTD₄ (10^–8 M), resulted in TxA₂ production in pg/10⁵ cells to the extent of: 67 ± 20, 571 ± 180, and 333 ± 60 respectively, as measured by radioimmunoassay of the stable metabolite TxB₂. Endothelial pretreatment with the LTD₄ receptor antagonist FPL55712 (10^–5 M) significantly blocked any LTB₄- or LTD₄-stimulated TxA₂ synthesis. Pretreatment with the TxA₂ synthetase inhibitor ketoconazole (10^–6 M) also prevented LTB₄ of LTD₄ stimulation of TxA₂. Preincubation with DMTU (10^–5 M), an hydroxyl radical scavenger, decreased LTB₄-induced release of TxA₂ (405 ± 40 and 366 ± 20, respectively). These findings suggest that LT may mediate their inflammatory actions in vascular beds by stimulation of Tx release from endothelial cells.     

Activation of the human neutrophil secretory process with 5(S),12(R)-dihydroxy-6, 14-cis-8,10-trans-eicosatetraenoic acid

Exposure of human neutrophils to 5(S),12(R) dihydroxy-6, 14-cis-8, 10-trans-eicosatetraenoic acid (leukotriene B₄, LTB₄) resulted in a time- and concentration- (10^–9-10^–6 M) dependent extracellular release of granule-associated lysozyme and myeloperoxidase (MPO). Enzyme extrusion was negligible if cells were not pretreated with cytochalasin B prior to exposure to LTB₄. A time-dependent deactivation of granule exocytosis was observed in neutrophils which were stimulated with LTB₄ prior to contact with cytochalasin B. LTB₄-induced enzyme release was markedly enhanced in the presence of extracellular calcium. Nevertheless, significant enzyme discharge occurred in the absence of extracellular calcium, and the percent of total activity released was not altered in the presence of EGTA. The calmodulin antagonist, trifluoperazine (TFP), and the intracellular calcium antagonist, 8-(N, N-dietliylamino)-octyl-(3,4,5-trimethoxy)benzoate hydrochloride (TMB-8), caused a dose-related inhibition of enzyme release from LTB₄-stimulated neutrophils. Degranulation was suppressed by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), and the sulfhydryl reagents iodoacetic acid (IA) andN-ethylmaleimide (NEM). Sodium cyanide was inactive. Two inhibitors of transmethylation, 3-deazaadenosine (3-DZA) and L-homocysteine thiolactone (HCTL), alone or in combination, had no effect on LTB₄-elicited degranulation. The protein synthesis inhibitor, cycloheximide, was inactive, Neutrophils pretreated with LTR₄ or 5(S), 12(R), 20-trihydroxy-6, 14-cis-8,10-trans-eicosatetraenoic acid (20-OH-LTB₄, an -oxidation metabolite of LTB₄) were desensitized to the subsequent exposure to LTB₄. Cross-desensitization was also demonstrated between LTB₄ and 20-OH-LTB₄. The stimulus specific nature of LTB₄-induced desensitization of neutrophil degranulation was demonstrated by the fact that cells exposed to 1-O-hexadecyl/octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) orN-formyl-methionyl-leucyl-phenylalanine (FMLP) were capable of inducing granule exocytosis from LTB₄-pretreated neutrophils. Enzyme release from LTB₄-treated cells was suppressed with the phospholipase inhibitor, 4-bromophenacyl bromide (4-BPB), the cyclooxygenase/lipoxygenase inhibitor, ETYA, and the 5-lipoxygenase inhibitor, U-60, 257. However, the cyclooxygenase inhibitor, flurbiprofen, exerted a weak suppressive effect on LTB₄-induced degranulation.     

Generation of leukotriene B4 by hemodialyzer membranes: A novel index of biocompatibility

Summary Leukotriene B₄ (LTB₄) plays an important role in acute and chronic inflammatory and hypersensitive reactions. We studied the time course of LTB₄ biosynthesis in whole blood in 18 patients with end-stage renal failure maintained on regular hemodialysis with two different membranes, cuprophane and polyacrylonitrile (AN 69). The basal levels of LTB₄ from dialysis patients did not differ significantly from a normal control group. Compared to predialytic values, the cuprophane membrane caused a maximal release of LTB₄ by a factor of about 4.5 (p<0.01) within the first 10 to 20 minutes. Thereafter the level fell and returned to baseline range at the end of the hemodialysis session. With the use of the AN 69 membrane no significant increase of LTB₄ could be demonstrated. The changes in LTB₄ concentration showed a close temporal correlation to the alterations in white blood cell count. We conclude that (1) LTB4 is a biologically important mediator of neutrophil activation during hemodialysis, and (2) LTB₄ may be a sensitive marker of biocompatibility in vivo.Abbreviations LTB₄ Leukotriene B₄ - 5-HETE 5-Hydroxyeicosatetraenoic acid     

SC-41930, a leukotriene B4 receptor antagonist,inhibits 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) binding to epidermal cells

SC-41930, 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxyl]-3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid, a potent leukotriene-B₄ (LTB₄) receptor antagonist, inhibitsin vivo 12-hydroxyei-cosatetraenoic acid (12-HETE)-induced neutrophil infiltration, suggesting a potential 12-HETE receptor antagonist effect, as well. Since 12-HETE is assumed to have a pathophysiological role in inflammatory skin diseases, and epidermal cells possess high affinity binding sites for 12(S)-HETE, we studied the effect of SC-41930 on 12(S)-HETE binding to the human epidermal cell line, SCL-II. SC-41930 antagonized the 12(S)-HETE binding to SCL-II cells with a Ki of 480 nM. This Ki value is similar to that obtained for the inhibition of LTB₄ binding to human neutrophils. Our results show that SC-41930, in addition to its LTB₄ receptor antagonist effect, exhibits 12-HETE receptor antagonist effect as well, and therefore may be of benefit in skin diseases with elevated 12-HETE levels.Dr. Kemény is on leave from the Department of Dermatology, Albert Szent-Györgyi Medical University, Szeged, Hungary, as a recipient of the Humboldt Fellowship, and his work was supported by the Alexander von Humboldt Foundation.     

Decreased histamine content and metabolism in mammary cancer tissue from C3H mice

Interleukin-8 (IL-8), is a potent activator of polymorphonuclear leukocyte (PMN) functions including chemotaxis, superoxide anion production, and enzyme release and it is also chemotactic for lymphocytes. Additionally, it has recently been shown that IL-8 stimulates the formation of 5-lipoxygenase (LO) products of arachidonic acid (AA) by human PMNs. The purpose of the present study was to determine whether IL-8 also might affect the formation of 15-LO products from AA. Purified PMNs in phosphate buffered saline were preincubated with and without exogenous AA (10^–5–10^–4 M) for 10 min. Then IL-8 was added in biologically relevant concentrations ranging from 0.1 to 100 ng/ml and incubation was carried out for 5 min at 37°C. Lipids were then extracted from supernatants, and eicosanoids were determined by quantitative RP-HPLC. Compared with unstimulated cells, IL-8 resulted in a dose dependent increase in both LTB₄ and 15-HETE (up to 125% and 40% at 100 ng/ml, respectively). This increase in eicosanoid formation required the presence of exogenous AA. These results indicate that IL-8 is both a potent stimulator of 5-LO activity and of 15-LO activity. LTB₄ can induce both inflammation and contribute to hyperproliferation in the skin. 15-HETE in contrast has the ability to inhibit the effects induced by LTB₄. Because IL-8 is able to stimulate both LTB₄ and 15-HETE formation, the effect of IL-8 as a putative regulator of inflammatory processes may be dependent on the relative stimulation of 5-LO and 15-LO.Part of this work has been presented at the Annual Meeting of the European Society for Dermatological Research (E.S.D.R.), Turin, Italy, June, 1990.     

Comparison of the effect between leukotriene B4 and leukotriene B5 on the induction of interleukin 1-like activity and calcium mobilizing activity in human blood monocytes

Leukotriene B₄ (LTB₄), one of 5-lipoxygenase (5-LO) products of arachidonic acid, was reported to be more potent than leukotriene B₅ (LTB₅), one of 5-LO products of eicosapentaenoic acid, in the activation of neutrophil functions through the differential potency between these leukotrienes in calcium mobilization. So we compared the effect of LTB₄ and LTB₅ on the induction of interleukin 1 (IL-1)-like activity and calcium mobilization in human blood monocytes. LTB₄ significantly augmented IL-1-like activity in monocytes, however, LTB₅ had no significant effect. Also LTB₄ was apparently more potent than LTB₅ in inducing calcium mobilization in fura-2-loaded monocytes. IL-1 production in monocytes was reported to be partly dependent on cytosolic free calcium. These results, therefore, may indicate that the different activity between LTB₄ and LTB₅ in the enhancement of IL-1-like activity could be partly ascribed to the different potency in inducing calcium mobilization between LTB₄ and LTB₅ in human blood monocytes.     

Interactions between synthesis of platelet-activating factor and leukotriene B4 in isolated human polymorphonuclear leukocytes

The aim of the present work was to study interactions between the synthesis of platelet-activating factor (PAF) and leukotriene B₄ (LTB₄) in human polymorphonuclear leukocytes (PMNs) in vitro. PAF, at nanomolar concentrations, stimulated calcium ionophore A23187-activated PMNs to release LTB₄ and 5-hydroxyeicosatetraenoic acid (5-HETE). This seems to be a receptor-mediated process as it was blocked by a PAF receptor antagonist WEB 2086 (IC₅₀ 6.6±3.9M). Moreover, LTB₄ stimulated the formation of PAF in activated PMNs. WEB 2086 did not, however, alter PMN migration towards either LTB₄ or the chemotactic peptide FMLP. This suggests that the enhancement of PAF synthesis in response to LTB₄ is a concomitant event rather than a mediating process in LTB₄-induced chemotactic movement of PMNs. These effects are implicated in the complex network of interactions between inflammatory mediators that results accumulation and activation of PMNs in the exacerbation of inflammatory processes.     

Release of granule proteins from human eosinophils stimulated with mast-cell mediators

Background It has been suggested that mast cells and eosinophils are major effector cells in the pathogenesis of allergic diseases. However, the interaction of these cells has not been thoroughly elucidated. We examined eosinophil cationic protein (ECP) release and cytosolic free calcium concentration ([Ca²⁺]) in human eosinophils induced by the major mast-cell mediators including cytokines. Methods Eosinophils from healthy donors were stimulated with the major mast-cell mediators for 20 min after preincubation with cytochalasin B for 10 min. ECP in supernatants was measured by radioimmunoassay. Moreover, t o examine changes of [Ca²⁺]i in eosinophils, Fura-2-loaded eosinophils were monitored for fluorescence changes after stimulus addition. Results Of the tested mediators (prostaglandin [PG]D₂, leukotriene (LT)B₄, platelet-activating factor (PAF), histamine, LTQ, and eosinophil chemotactic factor of anaphylaxis [ECF-A]), LTB₄ and PAF induced ECP release from eosinophils. Any cytokines produced by human mast cells, i.e., interleukin (IL)-4, IL-5, IL-8, tumor necrosis factor (TNF), or granulocyte-macrophage colony-stimulating factor (GM-CSF), did not induce ECP release in our system. ECP release triggered with LTB₄ and PAF occurred at concentrations of 10^?8-10^?6 M concentration-dependently. LTB4 and PAF also elicited a rise in [Ca²⁺]_i in eosinophils. Neither PGDj, histamine, nor LTC₄ induced ECP release, although they increased cytosolic calcium in eosinophils. Conclusions Of mast-cell mediators, LTB₄ and PAF induced eosinophil degranulation. The contribution of LTB4 and PAF from mast cells to eosinophil degranulation may be important in the pathogenesis of allergic inflammatory diseases.     

Intravenous anesthetic propofol suppresses leukotriene production in murine dendritic cells

Leukotrienes, divided into cysteinyl leukotrienes (CysLTs), which are important mediators of asthmatic responses, and leukotriene B4 (LTB₄), a chemotactic and chemokinetic agent for leukocytes, are potent lipid mediators generated from arachidonic acid by 5-lipoxygenase (5-LO). Leukotrienes are also considered to have immunoregulatory and pro-inflammatory actions. Propofol is an intravenous anesthetic widely used for anesthesia and sedation that is alleged to possess anti-inflammatory properties. The present study examined the effect of propofol on leukotriene production by dendritic cells (DC). In murine bone marrow-derived DC, propofol significantly suppressed CysLT and LTB₄ production after short-term stimulation with zymosan. The protein levels of cytosolic phospholipase A2 and 5-LO, or arachidonic acid release from plasma membranes, were not affected by the presence of propofol. Although zymosan treatment induced or enhanced the phosphorylation of ERK1/2, p-38 MAPK, and JNK, which presumably up-regulates the activity of 5-LO, the presence of propofol had no additional effect on the phosphorylation status of any of these MAPKs. Similarly, zymosan significantly increased the concentration of intracellular calcium, which is the most crucial activator of 5-LO, but no additional concentration changes were observed with the addition of propofol. Lastly, in an in-vitro cell-free ferrous oxidation-xylenol orange assay, propofol significantly inhibited the 5-LO activity of purified human recombinant 5-LO enzyme with an IC₅₀ of ~7.5 µM. Thus, propofol’s inhibition of 5-LO is not likely restricted to the circumstances surrounding the production of leukotrienes from DC, but applicable to other types of immune and non-immune cells that produce leukotrienes. The 5-LO-inhibiting activity of propofol may, at least in part, contribute to the well-known anti-inflammatory activity of propofol.     

Target-Effector-Cell Interactions in the Human Natural-Killer(NK)-System: Isolation of Target Structures

The existence of structures on NK-sensitive target cells selectively recognized by the effector cells have been postulated. To test this hypothesis, four selected human cell lines were investigated for target-cell proteins which could serve as specific ligands for the putative NK-cell receptor(s). NP-40 extracts from two highly NK-sensitive (K 562 and Molt-4) and two rather insensitive cell lines (HL-60 and Reh-6) were fractionated on SDS-polyacrylamide gels and tested for their ability to inhibit binding of effector to target cells as well as NK cytotoxicity. Three fractions with molecular weights (MW) of 200, 120 and 80 ± 10 KD isolated from K 562 cells were able to inhibit binding of large granular lymphocytes (LGL) to K 562. Of the other cell lines, Molt-4 and HL-60, both which were able to inhibit lysis of K 562 in a cold target inhibition assay, showed also two inhibitory fractions with MW 120 and 80 KD, whereas Reh-6, which is not able to compete with ⁵¹Cr-labelled K 562 in a cytotoxicity assay, lacked these structures. The 200, 120 and 80 KD fractions isolated from K 562 and the 120 and 80 KID fractions from Molt-4 and HL-60 were able to inhibit lysis of K 562 cells when added to the cytotoxicity assay. By adsorption/elution of radiolabelled K 562 extracts to/from LGL it was possible to detect an 80 KID target-cell surface protein which became preferentially bound by LGL-enriched but not by LGL-depleted lymphocyte preparations. Our results indicate the existence of target-cell proteins in NK-sensitive cell lines which serve as specific ligands for binding of NK cells. These target-cell structures of human cell lines differed from NK target structures described for mouse-NK-sensitive cell lines.     

LTD4 increases cytosolic free calcium and inositol phosphates in human neutrophils: inhibition by the novel LTD4 receptor antagonist,SR2640, and possible relation to modulation of chemotaxis

LTD₄ increased the level of free intracellular calcium ([Ca⁺⁺]_i) and stimulated the production of inositol phosphates (IP) in human polymorphonuclear neutrophils (PMN). Calcium was predominantly mobilized from intracellular pools. After a single stimulus, the cells were refractory to a second challenge with the same concentration of LTD₄, but the calcium response to LTB₄ was normal. The rise in [Ca⁺⁺]_i as well as the stimulated production of IP was inhibited by the novel LTD₄ antagonist, SR2640. SR2640 also abolished the attenuation by LTD₄ of LTB₄-directed PMN chemotaxis. The results suggest that human PMN contain specific LTD₄ receptor that trigger phosphatidyl inositol hydrolysis by activation of phospholipase C, leading to intracellular calcium mobilization, which may be involved in modulation of chemotaxis.     

Eicosanoid production in rheumatoid synovitis

Leukotriene B₄ (LTB₄) synthesis in rheumatoid synovitis was studied using peripheral and synovial fluid polymorphonuclear leukocytes (PMNs) and rheumatic synovial lining cells. No differences were found in LTB₄ synthesis between peripheral PMNs from healthy volunteers and rheumatoid arthritis patients. When peripheral and synovial PMNs from the same RA patient were compared, arachidonic acidinduced LTB₄ synthesis in synovial fluid PMNs was increased 1.7–7.2 fold, whereas the response to Ca ionophore A23187 stimulation was similar. This suggests 5-lipoxygenase stimulating factor(s) in inflamed joints. Rheumatic synovial lining cells in a primary cell culture produced small amounts of LTB₄, the concentrations being less than 0.1 per cent of those of prostaglandin E₂ (PGE₂). PGE₂ synthesis in synovial cells was increased when arachidonic acid or interleukin-1 was added to the culture, whereas LTB₄ production remained unaltered. The present results suggest that in inflamed joints LTB₄ originates mainly from PMNs whereas synovial lining cells are the source for PGE₂.     

LTB4 and BLT1 in inflammatory arthritis

Inflammatory arthritis, including rheumatoid arthritis (RA), is characterized by infiltration of inflammatory cells into the joints. Biological agents targeting TNF-α and IL-6 dramatically improve RA. However, some RA patients do not respond to current treatments and these broadly active upstream biological agents increase the risk of severe infection. Therefore, there remains a need for other effective and safe treatments for RA. Many studies have implicated that blockade of leukotriene B4 (LTB₄) and its high affinity receptor BLT1 dramatically suppress arthritis in animal models. In addition, levels of LTB₄ in serum, synovial fluid and synovial tissue are increased in RA patients compared to healthy donors or osteoarthritis patients. These data suggest that LTB₄ and BLT1 likely contribute to the pathogenesis of human RA. However, several clinical trials inhibiting BLT1 in RA were not successful. Our recent data revealed that LTB₄ is a key mediator in a complement, lipid, cytokine and chemokine cascade that first initiates and then sustains neutrophilic inflammation in inflammatory arthritis. These new mechanistic studies suggest novel ways to target the LTB₄-BLT1 pathway for the treatment of RA and other inflammatory diseases.