Cytochrome P-450 4F18 Is the Leukotriene B4 ω-1/ω-2 Hydroxylase in Mouse Polymorphonuclear Leukocytes

Leukotriene B4 (LTB4) is a potent chemoattractant for polymorphonuclear leukocytes (PMN) and other cells. Human PMN inactivate LTB4 by ω-oxidation catalyzed by cytochrome P-450 (CYP) 4F3A. The contribution of the enzymatic inactivation of LTB4 by CYP4Fs to down-regulating functional responses of cells to LTB4 is unknown. To elucidate the role of CYP4F-mediated inactivation of LTB4 in terminating the responses of PMN to LTB4 and to identify a target for future genetic studies in mice, we have identified the enzyme that catalyzes the ω-1 and ω-2 oxidation of LTB4 in mouse myeloid cells as CYP4F18. As determined by mass spectrometry, this enzyme catalyzes the conversion of LTB4 to 19-OH LTB4 and to a lesser extent 18-OH LTB4. Inhibition of CYP4F18 resulted in a marked increase in calcium flux and a 220% increase in the chemotactic response of mouse PMN to LTB4. CYP4F18 expression was induced in bone marrow-derived dendritic cells by bacterial lipopolysaccharide, a ligand for TLR4, and by poly(I·C), a ligand for TLR3. However, when bone marrow-derived myeloid dendritic cells trafficked to popliteal lymph nodes from paw pads, the expression of CYP4F18 was down-regulated. The results identify CYP4F18 as a critical protein in the regulation of LTB4 metabolism and functional responses in mouse PMN and identify it as the functional orthologue of human PMN CYP4F3A. Leukotriene B4 (LTB4) is a potent chemoattractant for polymorphonuclear leukocytes (PMN) and other cells. Human PMN inactivate LTB4 by ω-oxidation catalyzed by cytochrome P-450 (CYP) 4F3A. The contribution of the enzymatic inactivation of LTB4 by CYP4Fs to down-regulating functional responses of cells to LTB4 is unknown. To elucidate the role of CYP4F-mediated inactivation of LTB4 in terminating the responses of PMN to LTB4 and to identify a target for future genetic studies in mice, we have identified the enzyme that catalyzes the ω-1 and ω-2 oxidation of LTB4 in mouse myeloid cells as CYP4F18. As determined by mass spectrometry, this enzyme catalyzes the conversion of LTB4 to 19-OH LTB4 and to a lesser extent 18-OH LTB4. Inhibition of CYP4F18 resulted in a marked increase in calcium flux and a 220% increase in the chemotactic response of mouse PMN to LTB4. CYP4F18 expression was induced in bone marrow-derived dendritic cells by bacterial lipopolysaccharide, a ligand for TLR4, and by poly(I·C), a ligand for TLR3. However, when bone marrow-derived myeloid dendritic cells trafficked to popliteal lymph nodes from paw pads, the expression of CYP4F18 was down-regulated. The results identify CYP4F18 as a critical protein in the regulation of LTB4 metabolism and functional responses in mouse PMN and identify it as the functional orthologue of human PMN CYP4F3A. How polymorphonuclear leukocytes (PMN), 2The abbreviations used are: PMN, polymorphonuclear leukocyte(s); DC, dendritic cells; CYP, cytochrome P-450; LTB4, leukotriene B4; 17-ODYA, 17-octadecynoic acid; CFDA-SE, carboxyfluorescein diacetate, succinimidyl ester; PBS, phosphate-buffered saline; LPS, lipopolysaccharide; RT, reverse transcription; HPLC, high pressure liquid chromatography; RP, reversed phase; GC/MS, gas chromatography/mass spectrometry. macrophages, and dendritic cells (DC) control the initiation and amplification of innate and adaptive immune responses is a critical question, and the 5-lipoxygenase product of arachidonic acid metabolism leukotriene B4 (LTB4)is central to the amplification process. 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The second is receptor desensitization, which is based on G protein-coupled receptor kinases and β-arrestin (27Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar, 28Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1072) Google Scholar). The desensitization process has been elucidated in depth (27Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar, 28Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1072) Google Scholar), whereas the relative importance of ligand inactivation remains unknown. The major pathway for the metabolism and inactivation of LTB4 in human PMN is the uptake of extracellular LTB4, which is initially converted to 20-OH LTB4 by CYP4F3A (29Soberman R.J. Okita R.T. Fitzimmons B. Rokach J. Spur B. Austen K.F. J. Biol. 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In rat PMN, the initial ω-1 and ω-2 oxidation products of LTB4 are 19-OH and 18-OH LTB4 (35Powell W.S. Gravelle F. J. Biol. Chem. 1990; 265: 9131-9139Abstract Full Text PDF PubMed Google Scholar, 36Powell W.S. Gravelle F. J. Biol. Chem. 1989; 264: 5364-5369Abstract Full Text PDF PubMed Google Scholar). These products (and LTB4 itself) can be converted by a 12-hydroxydehydrogenase to their 11,12-dihydro products (35Powell W.S. Gravelle F. J. Biol. Chem. 1990; 265: 9131-9139Abstract Full Text PDF PubMed Google Scholar, 36Powell W.S. Gravelle F. J. Biol. Chem. 1989; 264: 5364-5369Abstract Full Text PDF PubMed Google Scholar). In metabolism studies of LTB4 introduced into humans intravenously, 19-OH and 18-OH LTB4 were detected as urinary metabolites (37Berry K.A. Borgeat P. Gosselin J. Flammand L. Murphy R.C. J. Biol. Chem. 2003; 278: 24449-24460Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 38Kumlin M. Falk J.R. Raud J. Harad Y. Dahlen S.R. Granstrom E. Biochem. Biohys. Res. Commun. 1990; 170: 23-29Crossref PubMed Scopus (22) Google Scholar); these products are not formed in human PMN. The enzymes that catalyze the ω-1 and ω-2 oxidation and the 12-hydroxydehydrogenase reactions of LTB4 in mouse leukocytes have not been identified (39Yokomizo T. Ogawa O. Uozumi N. Kume K. Izumi I. Shimizu T. J. Biol. Chem. 1996; 271: 2844-2850Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 40Yokomizo T. Izumi T. Takahashi T. Kasama T. Kobayashi Y. Sato F. Taketani Y. Shimizu T. J. Biol. Chem. 1993; 268: 18128-18135Abstract Full Text PDF PubMed Google Scholar, 41Wainright S.L. Powell W.S. J. Biol. Chem. 1991; 266: 20899-20906Abstract Full Text PDF PubMed Google Scholar). Whether there is a unique role for CYP4F metabolism in terminating cellular responses to LTB4 is also unknown. Based on these considerations and with the ultimate goal of understanding the role of CYP4Fs in terminating responses to LTB4 in vivo, we sought to identify the enzyme responsible for the ω-1 and ω-2 oxidation of LTB4 in mouse PMN and other leukocytes and to identify a functional role for CYP4F family members in the regulation of PMN responses to LTB4. We identified CYP4F18 as the mouse LTB4 ω-1 and ω-2 hydroxylase in mouse PMN and macrophages. Inhibition of CYP4F18 by the irreversible inhibitor of fatty acid and eicosanoid ω-hydroxylases 17-ODYA resulted in a 220% increase in the chemotactic response of PMN to LTB4 and a marked increase in calcium flux in response to LTB4. Inhibition of human PMN CYP4F3A also resulted in clear augmentation of calcium flux in response to LTB4. In addition, CYP4F18 expression was induced in bone marrow-derived DC by bacterial lipopolysaccharide, a ligand for TLR4 and poly(I·C), a ligand for TLR3. However, when DC trafficked to popliteal lymph nodes, the pattern of CYP4F18 expression was down-regulated relative to CYP4F15. The results identify ω-1 and ω-2 oxidation by CYP4F18 as a step in controlling the inactivation of functional responses to LTB4 in mouse PMN. Isolation of Mouse Cells and Tissues—9–12-week-old male and female Balb/c mice were used for experimentation and were euthanized with CO2 in accordance with the guidelines of the Massachusetts General Hospital/Partners Committee on Research Animal Care. Selected tissues (liver, kidney, spleen, thymus, heart, lung, brain, ovary, and bladder) were dissected for RNA analysis immediately after euthanization. Axillary, inguinal, and popliteal lymph nodes were dissected and pooled for analysis. Peripheral blood samples were drawn from the tail veins of mice prior to euthanization. Isolation of Peritoneal PMN and Macrophages—To generate peritoneal PMN, mice were injected intraperitoneally with 1 ml of sterile 0.9% sodium caseinate. After 18 h the mice were euthanized, and 9 ml of sterile PBS was injected into the peritoneal cavity to harvest PMN by peritoneal lavage. To isolate peritoneal macrophages, mice were injected intraperitoneally with 1 ml of sterile 3% sodium thioglycollate, and macrophages were harvested 72 h after injection by peritoneal lavage. In Vitro Differentiation and in Vivo Injection of Mouse DC—Immature mouse DC were obtained by a modification of the method of Inaba et al. (42Inaba K. Inaba N. Romani M. Aya H. Deguchi M. Ikehara S. Muramatsu S. Steinman R.M. J. Exp. Med. 1992; 176: 1693-1702Crossref PubMed Scopus (3331) Google Scholar, 43Boonstra A. Asselin-Paturel C. Gilliet M. Crain C. Trinchieri G. Liu Y.-J. O'Garra A. J. Exp. Med. 2003; 197: 101-109Crossref PubMed Scopus (483) Google Scholar). Bone marrow cells were isolated by flushing the femurs and tibiae of mice with RPMI 1640 containing 10% heat-inactivated fetal bovine serum. The cells were filtered through a spleen mesh, centrifuged at 1200 rpm for 5 min, and resuspended in ACK buffer (BioWhittaker) for 5 min to lyse red blood cells. The cells at this stage were used to analyze the expression of CYP4F18 in total bone marrow. The cells were then washed and cultured (5 × 105 cells/ml) in RPMI 1640 supplemented with 25 mm HEPES, pH 7.4, 10% fetal bovine serum, 1 mm sodium pyruvate, 2 mm β-mercaptoethanol, granulocyte macrophage colony stimulating factor (100 units/ml) and interleukin-4 (100 units/ml). On day 2 and 4, the media and nonadherent cells were replaced with fresh media. On day 6, nonadherent cells (DC) were removed and resuspended in fresh medium at the same cell density. Differentiation to DC was confirmed by analysis of CD11c expression by flow cytometry. For studies of DC activation in vitro, the cells were stimulated with 10–100 ng/ml LPS (Escherichia coli, 055:B5) or 25–50 μg/ml poly(I·C) (Amersham Biosciences) and analyzed for CD80, CD86, and CYP4F isoform expression after 24 h. To target DC to popliteal lymph nodes, immature bone marrow-derived DC were labeled for 5 min with 5 μm CFDA-SE (carboxyfluorescein diacetate, succinimidyl ester) dye from (Invitrogen). The cells were washed twice, and then 5 × 105 cells were mixed with 1 μg/ml LPS in a 10-μl volume and injected into the right paw pads of mice. The mice were euthanized 18 h after injection, and popliteal lymph node tissue was dissected, dissociated with 1000 IU/ml collagenase D (Roche Applied Science) for 2 × 1 h at 37 °C, and filtered through a spleen mesh (44Mempel R. Hendrickson S.E. von Andrian U. Nature. 2004; 427: 154-159Crossref PubMed Scopus (1356) Google Scholar). The cells were resuspended in ACK buffer (BioWhittaker) for 5 min, washed in PBS, and centrifuged on 35% bovine serum albumin gradients for 15 min at 7000 rpm, 4 °C. The cells were collected from the interface of the bovine serum albumin gradient, and CFDA-SE dye-stained cells were isolated by flow cytometry for RNA analysis. RNA Blot Analysis—A CYP4F18 cDNA probe was synthesized by labeling a 297-bp PCR product with [α-32P]dCTP by random priming; the PCR product was generated from mouse PMN cDNA with primers F18-2F and F18-6R (Table 1). PCR conditions were 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s for 30 cycles, and the product was purified using a Geneclean Spin kit (Qbiogene) prior to radiolabeling. A mouse multiple tissue expression array from BD Biosciences (Clontech) was hybridized with the 297-bp probe (2 ng/ml, ∼2 × 106 cpm/ng) for 6 h at 65 °C in 10 ml of ExpressHyb buffer. The array was washed in 2× SSC, 1% SDS at 65 °C five times for 20 min and then 0.1× SSC, 0.5% SDS at 55 °C twice for 20 min and was exposed to Kodak XAR film overnight at -70 °C with an intensifying screen. Hybridization signals were quantified with a PhosphorImager.TABLE 1Summary of primers cDNA positions are relative to the ATG initiation codon (A = +1)NameDirectioncDNAPositionSequence (5′ to 3′)F18-1FSenseCYP4F181–18CGACCATGTCACAGCTGAGCCTGF18-13RAntisenseCYP4F181575–1543TCACTGCGCGCCTGCGCTGAGCGGCTCCACTTTF18-2FSenseCYP4F18172–196AACTGGATCTTGGGACACCTAGGCCF18-6RAntisenseCYP4F18468–444ATGGAAGGCAGGTGTCAGCATGCGAIsoform-specific primers4F13FSenseCYP4F13105–124GGCCTGGATCTATGCCTTCT4F13RAntisenseCYP4F13286–267ACACAGGTCCAACCCAAGAG4F14FSenseCYP4F1455–74TGGAAGACCCTGCTACTGCT4F14RAntisenseCYP4F14222–203CAAGCCCTGCTCTGTAGGAG4F15FSenseCYP4F15196–215ATGATCACACCCACTGAGCA4F15RAntisenseCYP4F15359–340TTTAGGGCTACCGAAGCTGA4F16FSenseCYP4F1673–92GGAGTGGCTTCCTGGATTTT4F16RAntisenseCYP4F16316–297ATGCAGGGTCAACAATCCTC4F18FSenseCYP4F1893–112GGCTTACATCCTGACCCAGA4F18RAntisenseCYP4F18269–250CAGCAGCATGCATCCCTGAA Open table in a new tab Isoform-specific RT-PCR—RNA was extracted from freshly isolated mouse cells and tissues using Tri reagent (Sigma). In addition, total RNA from selected mouse tissues (liver, ovary, and smooth muscle) was purchased from BD Biosciences Clontech. Reverse transcription was performed with a cloned avian myeloblastosis virus first strand cDNA synthesis kit (Invitrogen). Primers summarized in Table 1 (listed under “isoform-specific primers”) were used to detect CYP4F13 (182-bp product), CYP4F14 (168-bp product), CYP4F15 (164-bp product), CYP4F16 (244-bp product), and CYP4F18 (177-bp product) by isoform-specific PCR. The same annealing temperature can be used for all primer sets, but different polymerases/buffer systems require independent optimization to determine the annealing temperature critical to achieve specificity. An annealing temperature of 66 °C was optimal for PCRs containing 1 unit of Taq polymerase and 10–100 ng of DNA in 10 mm Tris-HCl, pH 9, 50 mm KCl, 1.5 mm MgCl2, 400 nm each primer, and 200 μm dNTPs. The PCR conditions were 94 °C for 30 s, 66 °C for 30 s, and 72 °C for 30 s; 30 cycles were used unless stated otherwise. Semiquantitative PCR for CYP4F18 was performed by including Quantum RNA Classic 18 S internal standard primers (Ambion); 25 cycles of PCR was representative of the linear range, and the 18 S primers were combined with Competimers (Ambion) in a ratio of 3:7. Alternatively, glyceraldehyde-3-phosphate dehydrogenase RT-PCR Control Amplimers (BD Biosciences Clontech) were used as an internal control. PCR products were resolved on a 1% agarose gel, and the bands were quantified using image analysis software. Flow Cytometry—DC cultures were stained with allophycocyanin, percyphycoerythrin, or fluoroisothiocyanate-conjugated control IgGs or monoclonal antibodies directed to CD11c, CD80, and CD86 (BD Biosciences) to define immature and mature populations using a double or tri-color labeling. The samples were analyzed by flow cytometry using a FACScalibur instrument and the Cellquest program (BD Biosciences). Expression of CYP4F18 Protein—The full-length cDNA coding region of CYP4F18 was amplified from PMN cDNA by PCR using Pfu polymerase (Stratagene) with primers F18-1F (which places the sequence CGACC immediately upstream of the ATG initiation codon) and F18-13R (which comprises the last 33 bp inclusive of the TGA stop codon). The PCR conditions were 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 2 min for 30 cycles. A PCR product of 1580 bp was cloned into pCR2.1-TOPO vector (Invitrogen), and sequence analysis confirmed its identity with CYP4F18. The cDNA for CYP4F18 was subcloned into p51polORbp bicistronic baculovirus vector designed for coexpression with NADPH cytochrome P-450 reductase. Preparation of baculovirus, infection of Sf9 cells (at the Protein Expression Center at California Institute of Technology and at Gentest Corporation, Woburn, MA), and fractionation of cell extracts and microsomes was performed as previously described (32Christmas P. Jones J.P. Patten C.J. Rock D. Zheng Y. Cheng S.M. Weber B.M. Carlesso N. Scadden D.T. Rettie A.E. Soberman R.J. J. Biol. Chem. 2001; 276: 38166-38172Abstract Full Text Full Text PDF PubMed Google Scholar). Microsomes were adjusted to a concentration of 7.7 mg protein/ml (Lowry assay), and CYP4F18 expression was confirmed by Western blotting (31Christmas P. Ursino S.R. Fox J.W. Soberman R.J. J. Biol. Chem. 1999; 274: 21191-21199Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Immunofluorescent Microscopy—For indirect immunofluorescent staining of CYP4F18, DC were washed in PBS and cytospun onto slides. The cells were fixed in 4% paraformaldehyde in PBS for 30 min, incubated with 50 mm ammonium chloride for 10 min, and permeabilized with 0.1% Triton X-100 in PBS for 4 min. The cells were then blocked in 10% goat serum for 30 min and labeled with affinity-purified rabbit anti-CYP4F3 (10 μg/ml) for 1 h at room temperature. The affinity purified antibody to CYP4F3 has been described previously (32Christmas P. Jones J.P. Patten C.J. Rock D. Zheng Y. Cheng S.M. Weber B.M. Carlesso N. Scadden D.T. Rettie A.E. Soberman R.J. J. Biol. Chem. 2001; 276: 38166-38172Abstract Full Text Full Text PDF PubMed Google Scholar, 34Christmas P. Carlesso N. Shang H. Cheng S.-M. Weber B.M. Preffer F.I. Scadden D.T. Soberman R.J. J. Biol. Chem. 2003; 278: 25133-25142Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), and it reacts with both human (CYP4F3) and mouse (CYP4F18) proteins. The secondary antibody was fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Jackson Laboratories), used at a 1:250 dilution in PBS for 1 h at room temperature. Immunofluorescent microscopy was performed using a Nikon FXA photomicroscope and IP Spectrum (Scanalytics, Vienna, VA) acquisition analysis software. Analysis of LTB4 and AA Metabolites by RP-HPLC—A 100-μl reaction mixture containing 1 mm NADPH, 10 μl of Sf9 microsomes expressing CYP4F18, 30 μm LTB4, and 2 μCi of [3H8]LTB4 in 100 mm potassium phosphate buffer, pH 7.4, was incubated at 37 °C for 30 min. After the incubation, the reaction was stopped by adding 100 μl of ice-cold ethanol and centrifuged (10,000 × g) for 10 min. The supernatant was removed, and 1 ml of 100 mm potassium phosphate buffer, pH 7.4, and 1 drop of formic acid was added. The products were extracted (twice) using 1 ml 1:1 (v/v) hexane/ethyl acetate. After vortexing four times for 15 s, the samples were centrifuged at 135 × g for 5 min to separate the two layers. The organic layer was removed and taken to dryness under vacuum. Metabolites were purified by RP-HPLC using a Chromolith Performance RP-18e (4.6 × 100 mm) column. The reversed phase solvents used were 8.3 mm acetic acid adjusted to pH 5.7 with ammonium hydroxide (solvent A) and 65:35 acetonitrile/methanol (solvent B). The initial mobile phase was 25% solvent B, which was held for 3 min. This was followed by a linear gradient to 100% solvent B over 32 min. Initially, 5% of the hexane/ethyl acetate extract was introduced onto the RP-HPLC column, and the effluent was monitored using UV detection at 270 nm and on-line radioactivity. The remainder of the hexane/ethyl acetate extract was then introduced onto the RP-HPLC column, and 2 fractions/min were collected for GC/MS analysis. Gas Chromatography/Mass Spectrometry—The reversed phase HPLC fractions of interest were taken to dryness under vacuum and derivatized for GC/MS analysis by the addition of 50 μl of 5% N,N-diisopropylethylamine in acetonitrile and 50 μl of 5% pentafluorobenzyl bromide in acetonitrile. The samples were kept at room temperature for 30 min and evaporated under a stream of nitrogen. The samples were further derivatized with the addition of 50 μl of acetonitrile and 50 μlof bis(trimethylsilyl)trifluoroacetamide by incubating at 60 °C for 30 min followed by evaporation under nitrogen. The samples were reconstituted in 20 μl of acetonitrile and subjected to GC/MS analysis (35Powell W.S. Gravelle F. J. Biol. Chem. 1990; 265: 9131-9139Abstract Full Text PDF PubMed Google Scholar, 45Wheelan P. Zirrolli J.A. Murphy R.C. J. Am. Soc. Mass Spectrom. 1996; 7: 129-139Crossref PubMed Scopus (37) Google Scholar, 46Murphy R.C. Harper T.W. Gaskell S. Mass Spectrometry in Biomedical Research. John Wiley & Sons Ltd., Chichester, UK1986: 11-29Google Scholar, 47Murphy R.C. Snyder F. The Handbook of Lipid Research, Mass Spectrometry of Lipids. 7. Plenum Press, New York1993: 131-188Google Scholar). A gas chromatograph/mass spectrometer (Trace 2000; Thermo Finnigan, San Jose, CA) was used for the electron ionization analysis. The electron ionization spectra were obtained at an electron energy of 70 eV and provided structural information regarding the hydroxyl group position from fragmentation events adjacent to the trimethylsilyl ether positions. Calcium Flux and Chemotaxis—For cell migration assays, bone marrow cells were isolated from the femurs and tibias of mice by perfusion with sterile PBS (48Gerard N.P. Lu B. Liu P. Craig S. Fujiwara Okinaga Y.S. Gerard C. J. Biol. Chem. 2005; 280: 40996-41004Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). In some experiments they were further isolated by a discontinuous Percoll gradient at 500 × g for 30 min at room temperature (49Lowell C.A. Berton G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7580-7584Crossref PubMed Scopus (145) Google Scholar, 50Kruger J. Butler J.R. Cherapanov V. Dong Q. Ginzberg H. Govindarajan A. Grinstein S. Siminovitch K.A. Downey G.P. J. Immunol. 2000; 165: 5847-5859Crossref PubMed Scopus (69) Google Scholar). The three-step gradient was 55, 65, and 75% (v/v) Percoll in PBS. The mature PMN are recovered at the interface of the 65 and 75% fractions. PMN purity was >97% as determined morphometrically by Diff-Quik staining. The cells were fluorescently labeled with 5 μm calcein AM (Invitrogen), suspended in 20 mm HEPES, pH 7.5, 125 mm NaCl, 5 mm KCl, 1 mm MgCl2, 1 mm CaCl2, 0.5 mm glucose, 0.2% bovine serum albumin, at 1 × 107/ml (48Gerard N.P. Lu B. Liu P. Craig S. Fujiwara Okinaga Y.S. Gerard C. J. Biol. Chem. 2005; 280: 40996-41004Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The cells were also incubated with 30 μm 17-ODYA or buffer containing 0.05% Me2SO (controls) for 30 min and then tested for chemotactic activity in modified Boyden chambers. The cells (0.1 ml) were placed in the upper wells of 3-micron, 6.4-mm FluoroBlok filter inserts (Falcon) with 0.6 ml of buffer containing 1.0, 10, or 100 nm of LTB4 or 20-OH LTB4. The chambers were incubated at 37 °C for 45 min, and chemotaxis was determined by measuring the fluorescence intensity (excitation, 485 nm; emission, 535 nm) passing to the underside of the filter. The measurements were determined in quadruplicate for independent experiments. For calcium flux determination (51Gao J. Choe H. Bota D. Wright P.L. Gerard C. Gerard N.P. J. Biol. Chem. 2003; 39: 37902-37908Abstract Full Text Full Text PDF Scopus (43) Go