- Email: [email protected]
mass spectrometry
PROSTAGLANDINS
SYNTHESIS ALVEOLAR
R.K.
OF LEUKOTKIENE MACROPHAGES: MASS
J. Macl$ermot*, Knight , P.J.
C.l$. Cole
B AND PROSTANOIDS BY HUMAN ANA&S BY GAS CHROMATOGRAPHY/ SPECTROMETRY
Kelsey+,
, C.T.
K.A. Dollery*,
Waddell* R. R&hmond*, D.N. Landon & I.A.
*Department of Clinical PQarmacology, Royal Postgraduate London W12 OHS, UK, Host Dcfence Unit,+Department Cardiothoracic Institute, London SW3, UK, University Clinical Neurology, Institute of Neurology, London
Blair*
Medical School, of Medicine, Department of WCl, UK
ABSTRACT Human alveolar macrophages, obtained during diagnostic bronchoscopy , were maintained in monolayer culture. Challenge of these cells 095% purity) with 1.2 mglml zymosan A particles (opsonized with human into the serum) was followed by a rapid release of leukotriene B n = 4). medium, 7.28 ? 5.99 nglmg cell protein at 2 h (mean + S.04, was identified and measured by a novel technique Leukotriene B employing cap1*R ary column gas chromatography coupled to negative ion chemical ionization mass spectrometry . The release of thromboxane B2, enzyme N-acetyl-D-Dprostaglandins D2, E2, F2a and the lysosomal T hromboxane B was the most glucosaminidase was also measured. abundant metabolite of arachidonic acid released into the2 culture medium (65.2 + 14.8 nglmg cell protein 2 h after the addition of zymosan A, n = 4). and the the synthesis of thromboxane B2 was inhibited by >90% in 1 PM Na flurbiprofen. Inhibition of cyclooxygenase activity was accompanied by a 2-fold increase in leukotriene B4 synthesis. INTRODUCTION It is Leukotriene B4 (1) is a product of fatty acid Iipoxygenase. a putative mediator of inflammation that initiates leucocyte chemotaxis and increases vascular permeability in vivo when administered together Leukotriene B4 is with the vasodilator prostaglandin_(2-4). including synthesised widely by immunologica ?ly competent cells neutrophil and eosinophil polymorphonuclear leucocytes, monocytes and recently in rnacrophages (2). Leukotriene B4 has been demonstrated human alveolar macrophages (5). The synthesis and release of leukotrienes and other eicosanoids by macrophages is facilitated 9~ zymosan (6) , immunoglobulins G (7) and E (8,9), and the Ca ionophore A23187 (5,9). Macrophages also synthesise and release prostaglandins (10-15) and thromboxane A Their synthesis from arachidonic acid is (16). mediated throug h the cyclooxygenase pathway, and, like Iipoxygenase, the activity of this enzyme complex is limited by the release of arachidonic acid from the lipid pool by the action of phospholipase t2 The synthesis (17) or phospholipase C and a diglyceride lipase (18).
FEBRUARY
1984 VOL. 27 NO. 2
163
PROSTAGLANDINS
of cyclooxygenase products by macrophages is thus initiated by those stimuli that also facilitate the release of leukotriene B-4. programme designed to elucidate the role of As part of a arachidonic acid metabolism in human lung, we have examined the synthesis of eicosanoids by human macrophages in culture. A number of similar studies have been carried out previously in macrophages, but most have relied on the use of liquid scintillation counting tecl-+niques fqr the quantification and identification of the metabolites of H- or This method provides much useful C-arachidonic acid (S-8,10-14). and includes the potential for identifying all 20-carbon information, metabolites of arachidonic acid. However, the relationship of these results to prostaglandin (or thromboxane) release from endogenous substrate remains uncertain, particularly as prostaglandins are known to be released from multiple endogenous pools (13). Methods for the quantification of products derived from endogenous arachidonic acid substrate have included: bioassay of leukotrienes high performance liquid chromatography separated by and (9) radioimmunoassay and thromboxane B of prostaglandins (16) and leukotriene B4 (19). These techniques lack the . specl Y2IcIty of gas have been used as they are chromatography/mass spectrometry , but very sensitive and amenable to a large sample throughput. We have recently reported highly sensitive and specific methods for the analysis of prostanoids based on capillary column gas chromatography/negative ion chemical ionization mass spectrometry (20,21). We report the extension of this methodology for the analysis of leukotriene B4. In addition, we report data obtained using this technique on the synthesis of leukotriene B4 and prostanoids by human alveolar macrophages during phagocytosls of opsonized zymosan. MATERIALS
AND
METHODS
Human
alveolar macrophages were obtained during diagn-oscopy . Informed patient consent and local ethical After local anaesthetic with 2% committee approval were obtained. the airways were irrigated four times with 50 ml of (w/v) Iignocaine, 154 mM NaCl containing 0.283 PM NaHC03 pH 7.4 (solution A). Between 30% and 50% of &he irrigation fluid was recovered, and cell 10-90 x 10 cells. Cells were transported to the yields were solution A at 4OC. The cells were pelleted by laboratory in centrifugation at 150 g for 5 min and then washed 2 3Py suspvsion in 40 ml Dulbecco’s phosphate buffered saline (no Ca or Mg ions) (PBS) followed by centt$fugation at 150 g for 5 min. The cells were culture flasks (Falcon Labware) containing then plated in 25 cm Dulbecco’s modification of Eagle’s minimum essential medium (DMEM, Gibco Bio-Cult) with 10% (v/v) fetal calf serum (Gibco Bio-Cult) and 50 pglml gentamicin sulphate (Sigma London Chemical Co.). The flasks were maintained in a humidified atmosphere at 37’C containing 10% C02. After 16 h, non-adherent cells were removed by washing the plate 3 Experiments were performed in 5 ml of DMEM. times with 10 ml DMEM. Cells removed from the lung were pelleted by Microscopy. centrifugation at 150 g and fixed in 0.1 M cacodylate buffer pH 7.4 containing 3% (v/v) glutaraldehyde and 5 mM CaC12. The fixed cells were dehydrated in ethanol solutions of increasing sequentially
164
FEBRUARY
1984 VOL. 27 NO. 2
PROSTAGLANDINS
concentrations. They were then embedded in Epon 812 epoxy-resin (Shell Chemicals) and sectioned with a diamond knife. Plastic sections (1 urn) were stained with 1% (w/v) toluidine blue in 1% borax buffer Thin sections (60-70 nm) mounted and examined by light microscopy. saturated on 400 mesh copper grids were double stained with and an aqueous solution of 0.1% (w/v) uranylacetate in 100% methanol, lead citrate. by electron microscopy These sections were ex yined (80 Kv) at magnifications of 3.3-6.6 x 10 . Opsonized zymosan A. Lyophilized zymosan A from S. cerevisiae was obtained from Sigma London Ltd. A suspension of zymosan A (20 mg dry weight/ml) in PBS was boiled for 30 min. The particles were then washed 3 times in 10 ml PBS by resuspension and centrifugation at 3000 g for 5 min. The zymosan A particles (4 ml of 20 mglml in PBS) were opsonized with 2 ml pooled human serum by incubation at 37OC for 30 min. The opsonized zymosan A was finally washed 3 times as before with 10 ml PBS, and resuspended in PBS (20 ma/ml). Measurement of prosiaglandins and leukotriene B,, thromboxane B At the end of each timed’incubation of cells in DMEhl (5 ml), 3.5 rr?l’of DMEM was removed and added to 0.35 ml 0.5 M Tris-HCI buffer pH 7.4. Ty each sample was then a2dded 10 ng each of the folpwing standards: [ H4] prostaglandin F [ H lprostaglandin E and [ H ]6-oxo-prostaglandin F The sar@ies w&e centrifuged a3 3000 g 4or 5 min to remove \& zymosan A particles, and the supernatants frozen and stored at -2OOC. redistilled analytical grade solvents In the analysis of the samples, were used. Silica and uBondapak Cl8 reverse-phase Sep-Pak cartridges were supplied by Waters Assoc. (Inst.) Ltd. Sephadex LH-20 was obtained from Pharmacia Fine Chemicals. Methoxyamine hydrochloride (Eastman Co.) was recrystallized before use from ethanol containing about 1% (v/v) concentrated HCI. Pentafluorobenzyl (PFB) bromide (Fluorochem, UK) and bis-(trimethylsilyl)-trifluoroacetamide (Pierce Co.) were used without further purification. Prostaglandins, thromboxane B and the deuterated standards were kind gifts of the Upjohn Corn pans. Leukotriene B was3 a kind gift of Dr. J. Rokach lh,lSH-leukotriene B (30 Cilmmol) (Merck Frosst Canada Inc.). 5S,12Swas purchased from New England Nuclear, Boston, MA. dihydroxy-6,8,10,14-(E,Z,E,Z)-eicosatetraenoic 5S,l2Sacid, and 5S,l2R-dihydroxy-6,8,10,14(E,E,E,Z)-eicosatetraenoic acids were kind gifts of Dr. John Salmon. 8S,l5S-dihydroxy-5,9,11,13-(Z,E,Z,E)eicosatetraenoic and erythro-14,15-dihydroxy-5,8,10,12-(Z,Z,E,E)eicosatetraenoic acids Alan Brash and were kind gifts of Dr. Dr. John Murray. Extraction. Samples for analysis were thawed over 30 min at room temperature. The fluid was acidified to pH 3.5 with 2 M HCI and applied to a pre-washed reverse-phase C uBondapak Sep-Pak. The Sep-Pak was washed with 2 ml of distilldi water, and the eicosanoids eluted with 7 ml of ethyl acetate. This solution was applied to a straight phase silica Sep-Pak, and the ethyl acetate that eluted was discarded. The Sep-Pak was washed with 2 ml ethyl acetate and the eicosanoids eluted with 5 ml methanol. The methanol was evaporated under N2 and the residue transferred with a further 400 ul methanol to a small vial.
FEBRUARY
1984 VOL. 27 NO. 2
165
PROSTAGLANDINS
Derivatisation (a) Methoxlmation. The methanol was evaporated under N2 and with methoxyamine the dry extracts were treated 100 ul of hydrochloride in dry pyridine (5 mg/ml). The mixture was allowed to stand overnight at room temperature, and the pyridine then evaporated under N . (b)* Pentafluorobenzyl esterification. The crude residue from the methoximation step was dissolved In 36 ul acetonitrile. To this was added 10 ul 35% (v/v) PFB bromide acetonitrile and 10 ul in N ,N-diisopropylethylamine, and the solution heated to 40°C for 15 min. After cooling, the reagents were evaporated under N2, and the residue dissolved in 400 ul dichloromethane. The solution was applied to a short column of Sephadex LH-20 pre-swollen in dichloromethane and the derivatives eluted with a further 3 ml dichloromethane. (c) Trimethylsilylation. dichloromethane The solution was under N, residue evaporated the dissolved in 50 ul and his-(trimethylsilyl)-triflforoacetamide (bis-TMS-trifluoroacetamide) . After standing overnight at room temperature, the reagent was evaporated under dry N and the residue dissolved in n-dodecane ready for qas chromatoqraphic analysis. f mass spectrometric -Capillary caumn gas chromatography. Gas chromatography was carried out on a Finnigan 9600 with a glass-lined injector maintained at Injections were made in the
166
FEBRUARY
1984 VOL. 27 NO. 2
PROSTAGLANDINS
E 2 as the standard for prostaglandin leukotriey B , 12H lprostaglandin D2 and [ H f6-oxo-4prostaglandin F as the standard for thromboxane Furthir details of the prostai?andin analysis have been published B2’ previously (20.21). Measurement of N-acetyl+D-glucosaminidase 3.2.1.30) (EC zt$i;vied p;;;dmz;h;;2)employed was a modification of previously Volumes of 50 ul of the cell supernatants were incubated with 450 ul 4 mM p-nitrophenyl-N-acetyl+Dglucosaminide (Sigma, London, UK) in 40 mM citrate buffer pH 4.5 containing 5% (v/v) methanol. The incubation was allowed to proceed for 2-8 h, depending on the enzyme activity in the supernatant, and the reaction terminated by the addition of 1.5 ml 2 M glycine pH 10.7. The production of p-nitrophenol was monitored by the change in absorbance at 410 nm. The production of p-nitrophenol was linear with time and increasing enzyme concentration (to 5 mU / reaction volume) up to a final OD reading of 1.3 (1 unit of enzyme activity = 1 umol product/min). The protein content of the cultured macrophages was determined by a modification of the method of Lowry et al. (23). RESULTS The recovery of prostaglandins and thromboxane B2 through the extraction 2nd derivatization procedures was >85%. The recovery of H-leukotriene B4 carried through the extraction and 1 ng of derivatization procedure was 36% (S.D. = 5.1; n = 6). The negative ion mass spectrum (Fig. 1) of the bis-trimethylsilyl ether pentafluorobenzyl ester derivative of leukotriene Ba4 showed an intense ion at m/z -479 corresponding to [M-PFB] . This ion carried 20% of the tot2 Ton cur_rent. Fragment ions were also obse_rved at m/z -389 [M-PFB-TMSOH] and m/z -299 [M-PFB-(TMSOH) ] . The-i&s at -m/z -479 and m/z -389 were used for quanta 3 ative selected ion monitoring of lelkotriene B4. The limit of sensitivity was 2 pg on column. The retenjion time for leukotriene B was 7.31 min. and the retention time for [ H4lprostaglandin F20 was ‘4 .44 min under identical chromatographic conditrons. Quantification of leukotriene B was carried out by interpolation from linear regression curves of %uthentic standards in the range 0.1-10 ng. These were constructed by measurement of peak areas of the (M-PFB)ion at m/z -479 and the (M-PFB-TMSOH)ion at m/z -389 from leukotriene B -aEd determigng the ratio of these ionsthe (M-PFB)ion at -m?z -573 from ( H )-PGF . The area ratios were expressed as a percentage and a linelr reg&sion of log (concentration of leukotriene B in ng) against log (area ratio %) carried out. The equation of the tegression line for m/z -479 was y = exp (0.751 + 0.989 log x) r = 0.994. The equation ;f-the regression line for mlz -389 was y = exp (0.703 + 1.046 log x) r = 0.992. The Kovats Fecntion indices (24) were determined for a number of isomeric dihydroxy eicosatetraenoic acids using PFB-esters of saturated fatty acids as standards. The values obtained (Table 1) indicate that leukotriene B can be separated from these compounds on Sil-5 fused silica capillar 4 columns. Leukotriene B was baseline from separated to 5S,12S-dihydroxy-6,8,10,14-(r,Z,E,Z)-eicosatetraenoic acid, a selected
FEBRUARY
1984 VOL. 27 NO. 2
167
PROSTAGLANDINS
299
106
150
200
300
250
._.A
1
389
M-PFB -
ion chemical PFB-ester,
Table I. The Kovats eicosatetraenoic acids. Fatty
retention
ionisation mass spectrum ether derivative. -bis-TMS
indices
for
Acid
selected
Kovats
of
dihydroxy-
Retention
Index erythro-14,15-dihydroxy-5,8,10,12-(Z,Z,E,E)eicosatetraenoic 5S,12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-eicosatetraenoic 5S,12R-dihydroxy-6,8,10,14-(Z,E,E,Z)-eicosatetraenoic 8S,lSS-dihydroxy-5,9,11,13-(Z,E,Z,E)-eicosatetraenoic 5S,12S-dihydroxy-6,8,10,14-(E,E,E,Z)-eicosatetraenoic 5S,12R-dihydroxy-6,8,10,14-(E.E.E,Z)-eicosatetraenoic
168
2222 2229 2302 2312 2455 2455
FEBRUARY
1984 VOL. 27 NO. 2
PROSTAGLANDINS
ion current profile of the channel used to monitor m/z -479 is shown in Figure 2b. the ionization Under conditions - employed, little fragmentation of the 5S,lZS-dihydroxy fatty acid occurred. Thus, monitoring the mlz -389 ion a response could only be observed for leukotriene B (Figure 2a). The 5S.12S and 5S,lZR-dihydroxy6,8,10,14-(E,e,E,Z)-eicosatetraenoic acids co-eluted (Kovat’s indices 2455), however they were well resolved from leukotriene B4 in both the m/z -389 (Figure 2c) and m/z -479 channels (Figure 2d). -Examination by light>?roscopy of the cells obtained from human airways revealed that >95% of the cells present were a single morphological type. These were shown to be macrophages by electron microscopic examination of their ultrastructure. On a few occasions, the cells were heavily contaminated with desquamated epithelial cells, and on these occasions there was little or no adhesion of the macrophages to the culture flasks, These specimens were always discarded. Those specimens found suitable for these studies (about 4 out of every 5 samples) were plated as described in the Methods section. The plating efficiency varied between 55% and 90%, gnd the numbers gf adherent cells/flask varied between 0.5 x 10 and 2 x 10 . Microscopic examination of the adherent cells revealed that >98% were Adherent cells were examined for Trypan blue exclusion macrophages. before and after 5 h incubations in the absence or presence of zymosan A opsonized with human serum. The results showed that viability of the cells, as measured by this technique, was >90%. The non-adherent cells were mostly erythrocytes and lymphocytes. Cultured macrophages in replicate flasks were incubated for up to 5 h in the absence or presence of 1.2 mglml zymosan A opsonized with human serum. The release of prostaglandins D2, E2, F20, and into the culture medium in a typical experiment are thromboxane B shown in Fig 23 .A and B. A wide variation was observed in the concentrations of these eicosanoids, and no 6-oxo-prostaglandin Fla, the stable hydrolysis product of prostacyclin, was detected in any of the experiments. The release of all eicosanoids examined, with the exception of 6-oxo-prostaglandin Fla, was facilitated by opsonized zymosan A particles, although there was a significant release in the absence of zymosan A. The levels of prostaglandins released from macrophages were remarkably consistent from lung to lung, with the exception of prostaglandin D2. The results of eicosanoid release from cells obtained at 4 separate bronchoscopies were as follows (5 h after the addition of opsonized zymosan): prostaglandin E = 2.73 2 0.45 nglmg cell protein,prostaglandin F2 = 7.08 2 2.70 ng?mg cell protein, thromboxane B2 = 65.2 2 14.8 ng?mg cell protein and prostaglandin D = 24.5 + 37.4 nglmg cell protein (prostaglandin D2 range = 3.05 to Sd.4 nglmg cell protein; results show means + S. D. ) . In other experiments (data not presented), the release of these prostaglandins from alveolar macrophages was measured for times up to There was a gradual increase in prostaglandin and thromboxane 8 h. B2 concentration in the culture medium, with little tendency to plateau even at 8 h. In the experiment presented in Fig 3D, the culture medium was also analysed for its content of the lysosomal enzyme N-acetyl+D-glucosaminidase. The release of the enzyme from alveolar
FEBRUARY
1984 VOL. 27 NO. 2
169
PROSTAGLANDINS
macrophages opsonized unstimulated
was thus monitored The zymosan A. cells was increased
100- A
for 5 h in the absence or presence gradual release of the enzyme by the addition of zymosan A.
of in
- c
-
D
+=+Qo 1-
180
Scans Elution profiles of dihydroxyeicosatetraenoic acids Fig. 2. Results show from a capillary column gas chromatograph. (retention time = 7.31 the separation of mixtures of LTB SS,!‘tl2S-dihydroxy-6,8,10,14and min) (E,Z,E,Z)-eicosatetraenoic acid (retention time = 5.25 min) in 5S,12S and separation of A and the and B, 5S,12R-dihydroxy-6,8,10,14(E,E,E,Z)-eicosatetranoic acids (retention times both = 8.69 min) from LTB in C and D. These compounds were identified by selected ic?n monitoring at m/z -389 (A,C) or -m/z -479 (B,D). --
170
FEBRUARY
1984 VOL. 27 NO. 2
PROSTAGLANDINS
their capacity to also examined for release Cells were 3C) reveal a time course for the The results (Fig. leukotriene 6 . differs significantly from that observed BQ that release of le$kotriene prostaglandins, thromboxane and release of the for the B2 There was little or no release of N-acetyl+-D-glucosaminidase. leukotriene BG from cells cultured for 5 h in the absence of opsonized addition of zymosan increased the release of However, zymosan A. This result compares with the leukotriene B4 at least 20-fold at 2 h. 2-fold change after zymosan observed in the release of the
Hours D2 ( A A ), E2 B ( + * 1, are ’in F2% of2 N-acetyl-P-Dshown glucosaminidase ( + 0 )‘, is :~~wnr%a~e and the release of leukotriene B ( l 0 ) in C. Human alveolar macrophages the times shown in the absence (open were culture d for symbols) or presence (closed symbols) of 1.2 mg/ml opsonised zymosan A. Results are means (+ S.E.M.) of the concentrations of these compounds in the culture medium of The units in A, B, C are expressed as triplicate flasks. and in D the units are “enzyme activity” ng/mg cell protein, (nmol product/h) released per mg cell protein.
Wi
FEBRUARY
The
release ( n 0 and
of
), B
1984 VOL. 27 NO. 2
prostaglandins and thromboxane
171
PROSTAGLANDINS
Table -of human
2.
The effect of leukotriene B alveolar macorphag&.
sodium flurbiprofen thromboxane B2
Concentration Eicosanoid
Prostaglandin D2 Prostaglandin E2 Prostaglandin F 6-oxo-prostagla&in Thromboxane B2 Leukotriene B4
Control
Fla
on the synthesis and prostaglandins
(ng/mg
cell
and from
protein)
Flurbiprofen
4.88 3.01 3.18 <1.3 30.7 5.77
<1.3 <1.3 <1.3 <1.3 2.09 10.8
Alveolar macrophages were cultured and the release of eicosanoids in measured as described Methods. Flurbiprofen (1 uM) was added to the cells 2 min before the addition of zymosan A. The cell supernatants were harvested 3 h after the addition of opsonized zymosan A The results are the means of duplicate culture particles. flasks containing cells from one patient. prostaglandins, thromboxane B and the enzyme. Furthermore, no additional accumulation of leuko 2t riene B was observed in the culture medium after 2 h for times up to 8 h (da?a not presented). There was a significant variation in the levels of leukotriene B4 released from cells from 4 different patients (range = 1.15 to 15.5 nglmg cell protein) with a mean (2 S.D.) of 7.28 + 5.9? nglmg cell protein. The effect of sodium flurbiprofen on the synthesis and release of prostaglandins and thromboxane B2 was measured. A solution of 1 uM sodium flurbiprofen inhibited significantly synthesis of the prostaglandins D2, E2, F20 and thromboxane B2 (Table 2) in the presence of 1.2 mg/ml opsonized zymosan A. The synthesis and release of leukotriene B however, was increased 2-fold. The inhibition by sodium flurbiprofen in the of thromboxane B2 synt ‘h’esis and release was selected presence of opsonized zymosan A compared at concentrations of sodium flurbiprofen (Fig. 4). The results reveal a concentration for 50% inhibition by sodium flurbiprofen of 30 nM. In other experiments (data not presented), the effect of sodium flurbiprofen (100 uM) was examined on the release of N-acetyl-BD-glucosaminidase in the absence or presence of 1.2 mglml of opsonized zymosan A. There was, however, no effect of sodium flurbiprofen on basal or zymosan-stimulated enzyme release over a period of 5 h. DISCUSSION Previous methods for the analysis of endogenous leukotriene B4 have relied on the use of radioimmunoassay (19) or high performance liquid chromatography (25). We have examined the use of gas chromatography/mass spectrometry for the quantitative analysis of leukotriene B4. The technique should provide a method with a degree
172
FEBRUARY
1984 VOL. 27 NO. 2
PROSTAGLANDINS
[Na flurbiprofen]
M
4. The effect of sodium flurbiprofen on the *sis and release of thromboxane 62. The results were obtained by analysis of cell supernatants (duplicate flasks at each concentration) harvested 3 h after the addition of opsonized zymosan A particles. Cells were cultured in the absence or presence of flurbiprofen at the concentrations shown, and in the experiment, flurbiprofen was added to the cells 2 min before the addition of zymosan A.
of specificity that cannot be attained with either high performance liquid chromatography or radioimmunoassay. Electron impact mass spectrometry causes extensive fragmentation of the leukotriene molecule (26) so that selected ion monitoring techniques required for gas chromatography-mass relatively spectrometric analysis would be insensitive. However, it has been reported recently that negative ion chemical ionization mass spectrometry can be used for the efficient ionization of prostanoids (27). The technique relies on thermal electron capture in the gas phase, and the process is facilitated by prior conversion of the prostanoid carboxyl group to the PFB ester. Paradoxically, once the electron capture has taken place, the PFB moiety is lost and the stabilized carboxylate anion is formed. These
FEBRUARY
1984 VOL. 27 NO. 2
173
PROSTAGLANDINS
[M-PFB]ions are ideal for specific and sensitive quantitative analysis which suggested that the same technique of prostaglandins (20,21), could be used for the analysis of leukotriene B . was converted to its PBk ester bis-trimethylsilyl Leukotriene B ionization and subjec t ed to negative ion chemical mass ether It had similar characteristics to spectrometric analysis. the prostanoids although it showed a greater tendency to fragment Selected ion monitoring analysis of the [M-PFB] ion (Fig. 1). indicated that the limit of sensitivity for leukotriene B4 was 2 pg on A quantitative assay was designed in yhlch two ions, column. [M-PFE]and [M-PBF-TMSOH] , were monitored and [ H4lprostaglandin as the internal standard for quantitative analysis. F 2a. was employed The analysis chromatography/mass of leukotriene B4 by gas spectrometry is complicated by the possibility of interference from an isomeric dihydroxyeicosatetraenoic acid. If the specific hydrolase became enzyme which converts leukotriene A to leukotriene B saturated then aqueous hydrolysis wou l4d result in the for!nation of 5S,12S and 5S,12R-dihydroxy-C,8,10,14-(E,E,E,Z)-eicosatetraenoic acids The retention data in the literature suggests that these two (28). compounds (C values 24.8 as methyl esters) (29) would separate from There are no leukotriene B4 (C value 23.6 as methyl ester) (29,30). reported retention times for the corresponding PFB-esters so these were determined, and the Kovats Indices (24) calculated by interpolation from appropriate PFB-esters of saturated fatty acid standards (Table 1). no possibility of These data clearly established that there was interference in the assay from the aqueous hydrolysis products of The other possible interference was from the double leukotriene A . Iipoxygenase %roduct of arachidonic acid, 5S,12S-dihydroxy-6,8,10,14(E,Z,E,Z)-eicosatetraenoic acid (30.31). The retention data in the literature on this compound is equivocal. Three studies report C values on the methyl ester of 22.4 (1% SE-30) (31), 23.6 (1% SE-30) (30) and 23.7 (1% SP 2100) (32) respectively. It was noted in one report that the gas chromatography properties of the compound were poor (32). A similar observation was made previously on mono-hydroxy fatty acids in which there was a similar E,Z geometry at Perhaps the poor chromatographic C-6 and C-8 respectively (33). characteristics of the compound account for the discrepancy in the retention data. The PFB-ester also appeared to have poor characteristics although baseline resolution could be obtained between the double lipoxygenase B4 (Figure 2b). In addition, the product and leukotriene Under the fragmentation pattern of the molecule was quite different. -389 there ion at ionization conditions was no employed m/z to (M-PFB-TMSOH)(Figure 2a). This -iOn was corresponding employed for quantitative selected ion monitoring analysis and so there could be no possibility of interference in the assay for leukotriene B4 acid. 5S,12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-eicosatetraenoic by Finally, the Kovats indices were determined for two other isomeric 8S,15S-dihydroxy-5,9,11,13-(Z,E,Z,E)-eicosatetraenoic and acids, erythro-14,15-dihydroxy-5,8,10,12-(Z,Z,E,E)-eicosatetraenoic acids to show that they could not interfere in the assay (Table 1). the experimental design was similar in In the handling of cells, By maintenance many respects to methods described previously (10).
174
FEBRUARY
1984 VOL. 27 NO. 2
PROSTAGLANDINS
of the cells in monolayer culture, non-viable macrophages, erythrocytes and lymphocytes were removed from the culture flask as non-adherent cells in the washings. The time courses for the release of prostaglandins D E2, F and the lysosomal enzyme N-acetyl+-Dglucosaminidase *were SI‘I#?I ar, with a gradual increase in their concentrations in the culture medium, and little tendency to plateau over 8 h. In this respect these results are similar to those obtained with mouse peritoneal (10) and rabbit alveolar macrophages (11,12). The release of lysosomal enzymes accompanies phagocytosis, and is probably initiated by the fusion of the internalized ‘or pinocytic vesicle with a lysosome. Whether any benefit to the host accompanies the in vivo release of lysosomal hydrolases from macrophages into tt% extracellular environment is uncertain. The finding that inhibition of the cyclooxygenase activity (Table 2) with flurbiprofen had no effect on the release of the acid hydrolase N-acetyl+-D-glucosaminidase has been observed in similar experiments with animal macrophages (10,ll). There is an extensive literature on the different prostaglandins and their relative proportions released from macrophages under resting conditions or after stimulation (1 O-l 6.34). There is some consistency in the results, but differences have been observed between species and even among macrophages from different anatomical sites of the same species (34). The relative concentrations of arachidonic acid metabolites released from macrophages are altered only slightly in stimulated cells when compared to those in a resting state (34). (7,8,10-14,34), F Significant concentrations of prostaglandins E (ll-14,34), D2 (ll-14), C-0x0-Fl (8,10-14,16,3& and thromboxane 6” (16,34) are released from macro&ages of many species, and in moss reports prostaglandins E2, C-0x0-Flu and thromboxane B2 are the most abundant. Early reports of significant synthesis and release of prostaglandins El and F,o are not now generally accepted, and are probably the consequence of problems with analysis not appreciated at the time. In the present communication data are presented for the release of prostaglandins generated from endogenous substrate, thus permitting quantitative measurement of the total prostaglandins released in vitro Inspection of the results reveals from human alveolar macrophages. was the most abundant metabolite of arachidonic that thromboxane B acid, although sign1 .3 icant, but variable, levels of prostaglandin D2 were also observed. Prostaglandins E2 and F were also released at lower concentrations, but no C-oxo-prostaglan C!” In F was detected in the The absence of I%-oxo-prostaglandin F supernatants of these cells. in human alveolar macrophages is similar to that observed in alveoli? macrophages of mice (34), and it is intriguing to note that in the same lungs report, mouse pulmonary macrophages obtained from chopped released (rather than by airway lavage) synthesised and 6-oxo-prostaglandin F in response to zymosan. Human alveola r’” are known to macrophages synthesise leukotriene B4 (S), and in the present report quantitative measurement of its release in vitro has been provided for the first time. In based on capillary column gas addition, its physico-chemical properties, chromatography and negative ion chemical ionization mass spectrometry have identified this product with great certainty. The original report (5) identified leukotriene B4 on the basis of its chromatographic and UV
FEBRUARY
1984 VOL. 27 NO. 2
175
PROSTAGLANDINS
spectral properties. In addition, a bio-assay employing the neutrophil polymorphonuclear leucocyte chemotactic response demonstrated similar biological activities to authentic leukotriene B4. There was little or no release of leukotriene B4 from resting macrophages and addition of opsonized zymosan A particles resulted in an apparent more rapid release of leukotriene B than the cyclooxygenase products, with no additional release after tlhe first 2 h. However, the possibility that leukotriene B is metabolised in this system cannot be discounted. There w$s little or no synthesis of thromboxane B , or any of the prostaglandins measured, in the presence of 1 uM sod&m flurbiprofen, a potent inhibitor of cyclooxygenase activity Both the (35). cyclooxygenase and I ipoxygenase enzymatic pathways represent minor metabolite pathways for clearance of free arachidonic acid, the major portion being re-esterified and incorporated into membrane lipid . Nevertheless, inhibition of with 1 uM cyclooxygenase sodium flurbiprofen (Table 2) increased nearly 2-fold the synthesis and release of the lipoxygenase product leukotriene B4. The significance of this observation in relation to the regulation of lipoxygenase activity remains obscure, but may simply reflect an increase in substrate availability. In conclusion, the results provide quantitative measurement of leukotriene B thromboxane B and seve ra I prostaglandins from endogenous su b strate of human a 12 veolar macrophages cultured in vitro. The analyses were based on capillary column chromatography coupledo negative ion chemical ionization mass spectrometry. ACKNOWLEDGEMENTS Financial support Research Council (UK)
from the Wellcome Trust is gratefully acknowledged.
(UK)
and
the
Medical
REFERENCES
1.
Borgeat, P., and B. Samuelsson. acid by rabbit polymorphonuclear 1979. -254: 2643,
3.
Cunningham, F.M., H. R. Carter, M.J.H. Smith, A.W. Ford-Hutchinson and M.A. Bray. Aggregation of polymorphonuclear leucocytes (PMNs) by leukotriene B : Effects of cyclooxygenase products and metabolic inhibitors. A!gents and Actions 583, -11: 1981.
4.
Smith, M.J.H., Leukotriene B Pharmacol. -32:
176
A.W. a potential 517, 1980.
Fels, A.O.S., N.A. Z.A. Cohn, and W.A. leukotriene B4. Proc.
activities
of
leukotriene
of arachidonic Biol. Chem.
Smith, Actions
:
Biological 1981.
J.
2.
5.
M.J.H. -11: 571,
Transformation leukocytes.
Ford-Hutchinson, mediator of
B4.
and inflammation.
Agents
M.A. J.
and
Bray. Pharm.
Pawlowski, E.B. Cramer, T.K.C. King, Smith. Human alveolar macrophages produce Natl. Acad. Sci. USA -79: 7866, 1982.
FEBRUARY
1984 VOL. 27 NO. 2
PROSTAGLANDINS
6.
Rouzer, C.A., W.A. Scott, Z.A. Cohn, P. Blackburn, and J.M. Manning. Mouse peritoneal macrophages release leukotriene C in response to phagocytic stimulus. Proc. Natl. Acad. Sci. USA -77: 4928, 1980.
7.
Rouzer, C.A., Prostaglandin receptor-ligand 1980.
8.
Rouzer, C.A., W.A. Scott, A.L. Hamill, F-J. Liu, D.H. Katz, Z.A. Cohn. Secretion of leukotriene C and other arachidonic metabolites by macrophages challenged with immunoglobulin immune complexes. J. Exp. Med. -156: 1077, 1982.
9.
Rankin, Brashler, C4 from
10.
Bonney, R.J., P.D. Wightman, P. Davies, S.J. Sadowski F.A. Kuehl, and J.L. Humes. Regulation of prostaglandin synthesis and of the selective release of lysosmal hydrolases by mouse peritoneal macrophages. Biochem. J. -176: 433, 1978.
11.
Hsueh, W., prostaglandin phagocytosis 1979.
12.
Hsueh, W., F. Gonzalez-Crussi, synthesis in different phases of Nature -283: 80, 1980.
13.
Hsueh, W., U. Desai, F. Gonzalez-Crussi, R. Lamb, and A. Two phospholipase pools prostaglandin for synthesis macrophages. Nature 1980. -290: 710,
14.
Hsueh, W., R. Lamb, and F. Gonzalez-Crussi. phospholipase A2 activity and prostaglandin biosynthesis calmette-guerin-activated alveolar macrophages. Biochim. Acta. 1982. -710: 406,
15.
Stenson, W.F., and C.W. and immunity. J. Immunol.
16.
Cook, J.A., prostacyclin macrophages.
17.
Flower, R.J., phospholipase-A2 1976. -25: 285,
FEBRUARY
W.A. synthesis interaction.
Scott, .I. Kempe, by macrophages Proc Natl. Acad.
J.A., M. Hitchcock, and P.W. Askenase. alveolar macrophages.
C. Kuhn, secretion and lysosomal
and Z.A. Cohn. requires a specific Sci. USA -77: 4279,
W. Merrill, M.K. Bach, J.R. IgE-dependent release of leukotriene Nature -297: 329, 1982.
and P. Needleman. Relationship alveolar by rabbit macrophages enzyme release. Biochem. J. -184:
and E. Hanneman. phagocytosis in lung
Parker. -125: 1,
Prostaglandins, 1980.
1984 VOL. 27 NO. 2
of to 345,
Prostaglandin macrophages.
Chu. in
Decreased in bacillus Biophys.
macrophages,
W.C. Wise, and P.V. Halushka. Thromboxane produced by lipopolysaccharide-stimulated J. Reticuloendothel. Sot. -30: 445, 1981. and G.J. in prostaglandin
and acid E
A and pet-1.?oneal
Blackwell. The importance of biosynthesis. Biochem. Pharmac.
177
PROSTAGLANDINS
18.
Bell, R.L., D.A. Kennerly, N. Stanford, and P.W. Majerus. a pathway for arachidonate release from human Diglyceride lipas?: platelets. Proc. Natl. Acad. Sci. USA -76: 3238, 1979.
19.
Salmon, J.A., radioimmunoassay
P.M. Simmons, for leukotriene B4.
K.A., J. Wellby, gas chromatography Mass Spectrom. -10:
and R.M.J. Prostaglandins
and I.A. Blair. mass spectrometry 83, 1983.
-24:
20.
Waddell, column Biomed.
21.
Waddell, K.A., C. Robinson, M.A. Orchard, Blair. Quantitative analysis C.T. Dollery, and I.A. acid metabolites in biological fluids using GCINICIMS. Spectrom. Ion Phys. 1983. -48: 233,
22.
Schwartz, L.B., K.F. Austen, and S.I. Wasserman. release of p-hexosaminidase and 8-glucaronidase from serosal mast cells. J. Immunol. 1979. -123: 1445,
23.
Lowry, O.H., N.J. Protein measurement 1951. -193: 265,
24.
Wehrli, A., charakterisierung 2709, 1959.
and organic
25.
Mathews, W.R., leukotrienes by Biochem. 96, -lib:
J. Rokach, high-pressure 1981.
26.
Ford-Hutchinson, A.W., M.A. Bray, Leukotriene B, and M.J.H. Smith. aggregating substance released leukocytes. Nature -286: 264, 1980.
Rosebrough, with the folin
E.
28.
Maycock, A.L., Jr. Leukotriene cell-free system 1982.
29.
and B. Samuelsson. Borgeat, P., polymorphonuclear leukocytes. hydroxylated compounds. J. Biol.
178
S.E. Barrow, Prostacyclin is 23: 579, 1982. -
and
Blair, I.A., C.T. Dollery. Prostaglandins
Barrow, S.E. of arachidonic Int. J. Mass
Immunologic purified rat
Gas-chromatographische Helv. Chim. Acta
R.C. liquid
K.A. not a
M.S. Anderson, Preparation A4: to leukotriene
Combined capillary of prostanoids.
A.L. Farr, and P.J. Randall. phenol reagent. J. Biol. Chem.
Kovats. verbindungen.
27.
Palmer. A 225, 1982.
a
-42:
Murphy. Analysis of Anal. chromatography.
M.V. Doig, M. E. Shipley, potent chemokinetic and polymorphonuclear from
Waddell, circulating
P.J. Lewis, hormone in
and man.
D.M. De Sousa, and F.A. and enzymatic conversion B4. J. Biol. them. -257:
Kuehl, in a 13911,
Metabolism of arachidonic Structural analysis of Chem. 1979. -254: 7865,
acid in novel
FEBRUARY
1984 VOL. 27 NO. 2
PROSTAGLANDINS
P.,
S. of arachidonic
Transformation and
Med.
557,
P. acid
Vallerand, and in leukocytes.
P. Isolation
and
1981.
G.
31.
Lindgren, .A., novel hydroxylated polymorphonuclear
32.
Maas, R.L., A.R. Brash. the lo-carbon A,, by human
33.
Boeynaems, J.M., and W.C. Hubbard. Preparation, purification, characterization and assay of hydroxyand hydroperoxyeicosatetraenoic acids. In: Prostaglandins, prostacyclin and thromboxane measurement. Boeynaems, J.M. and Eds Herman, A.G., Nijhoff, The Hague, p. 167, 1980.
34.
Rouzer, Synthesis by mouse
35.
Cockbill, S.R., the abilities of to inhibit the human blood
Editor: Received: Accepted:
FEBRUARY
Hansson, and B. Samuelsson. Formation of eicosatetraenoic acids in preparations of human leukocytes. FEBS Lett. -128: 329, 1981.
C.D. Ingram, D.F. Taber, J.A. Oates, and Stereospecific removal of the D hydrogen atom at of arachidonic acid in the biosyn Fzhesis of leukotriene leukocytes. J. Biol. Chem. 1982. -257: 13515,
C.A., W.A. Scott, of leukotriene C and pulmonary macrophages.
Elisabeth 6-22-33 12-20-83
A.L. other
Hamill, and Z.A. Cohn. arachidonic acid metabolites J . Exp. Med. -155: 720, 1982.
S Heptinstall, and P.M. Taylor. A comparison of flurbiprofen and indomethacin acetylsalicylic acid, release reaction and prostaglandin synthesis in Pharmac. 73, 1979. platelets. Br. J. -67:
Granstrom
1984 VOL. 27 NO. 2
179
SYNTHESIS ALVEOLAR
R.K.
OF LEUKOTKIENE MACROPHAGES: MASS
J. Macl$ermot*, Knight , P.J.
C.l$. Cole
B AND PROSTANOIDS BY HUMAN ANA&S BY GAS CHROMATOGRAPHY/ SPECTROMETRY
Kelsey+,
, C.T.
K.A. Dollery*,
Waddell* R. R&hmond*, D.N. Landon & I.A.
*Department of Clinical PQarmacology, Royal Postgraduate London W12 OHS, UK, Host Dcfence Unit,+Department Cardiothoracic Institute, London SW3, UK, University Clinical Neurology, Institute of Neurology, London
Blair*
Medical School, of Medicine, Department of WCl, UK
ABSTRACT Human alveolar macrophages, obtained during diagnostic bronchoscopy , were maintained in monolayer culture. Challenge of these cells 095% purity) with 1.2 mglml zymosan A particles (opsonized with human into the serum) was followed by a rapid release of leukotriene B n = 4). medium, 7.28 ? 5.99 nglmg cell protein at 2 h (mean + S.04, was identified and measured by a novel technique Leukotriene B employing cap1*R ary column gas chromatography coupled to negative ion chemical ionization mass spectrometry . The release of thromboxane B2, enzyme N-acetyl-D-Dprostaglandins D2, E2, F2a and the lysosomal T hromboxane B was the most glucosaminidase was also measured. abundant metabolite of arachidonic acid released into the2 culture medium (65.2 + 14.8 nglmg cell protein 2 h after the addition of zymosan A, n = 4). and the the synthesis of thromboxane B2 was inhibited by >90% in 1 PM Na flurbiprofen. Inhibition of cyclooxygenase activity was accompanied by a 2-fold increase in leukotriene B4 synthesis. INTRODUCTION It is Leukotriene B4 (1) is a product of fatty acid Iipoxygenase. a putative mediator of inflammation that initiates leucocyte chemotaxis and increases vascular permeability in vivo when administered together Leukotriene B4 is with the vasodilator prostaglandin_(2-4). including synthesised widely by immunologica ?ly competent cells neutrophil and eosinophil polymorphonuclear leucocytes, monocytes and recently in rnacrophages (2). Leukotriene B4 has been demonstrated human alveolar macrophages (5). The synthesis and release of leukotrienes and other eicosanoids by macrophages is facilitated 9~ zymosan (6) , immunoglobulins G (7) and E (8,9), and the Ca ionophore A23187 (5,9). Macrophages also synthesise and release prostaglandins (10-15) and thromboxane A Their synthesis from arachidonic acid is (16). mediated throug h the cyclooxygenase pathway, and, like Iipoxygenase, the activity of this enzyme complex is limited by the release of arachidonic acid from the lipid pool by the action of phospholipase t2 The synthesis (17) or phospholipase C and a diglyceride lipase (18).
FEBRUARY
1984 VOL. 27 NO. 2
163
PROSTAGLANDINS
of cyclooxygenase products by macrophages is thus initiated by those stimuli that also facilitate the release of leukotriene B-4. programme designed to elucidate the role of As part of a arachidonic acid metabolism in human lung, we have examined the synthesis of eicosanoids by human macrophages in culture. A number of similar studies have been carried out previously in macrophages, but most have relied on the use of liquid scintillation counting tecl-+niques fqr the quantification and identification of the metabolites of H- or This method provides much useful C-arachidonic acid (S-8,10-14). and includes the potential for identifying all 20-carbon information, metabolites of arachidonic acid. However, the relationship of these results to prostaglandin (or thromboxane) release from endogenous substrate remains uncertain, particularly as prostaglandins are known to be released from multiple endogenous pools (13). Methods for the quantification of products derived from endogenous arachidonic acid substrate have included: bioassay of leukotrienes high performance liquid chromatography separated by and (9) radioimmunoassay and thromboxane B of prostaglandins (16) and leukotriene B4 (19). These techniques lack the . specl Y2IcIty of gas have been used as they are chromatography/mass spectrometry , but very sensitive and amenable to a large sample throughput. We have recently reported highly sensitive and specific methods for the analysis of prostanoids based on capillary column gas chromatography/negative ion chemical ionization mass spectrometry (20,21). We report the extension of this methodology for the analysis of leukotriene B4. In addition, we report data obtained using this technique on the synthesis of leukotriene B4 and prostanoids by human alveolar macrophages during phagocytosls of opsonized zymosan. MATERIALS
AND
METHODS
Human
alveolar macrophages were obtained during diagn-oscopy . Informed patient consent and local ethical After local anaesthetic with 2% committee approval were obtained. the airways were irrigated four times with 50 ml of (w/v) Iignocaine, 154 mM NaCl containing 0.283 PM NaHC03 pH 7.4 (solution A). Between 30% and 50% of &he irrigation fluid was recovered, and cell 10-90 x 10 cells. Cells were transported to the yields were solution A at 4OC. The cells were pelleted by laboratory in centrifugation at 150 g for 5 min and then washed 2 3Py suspvsion in 40 ml Dulbecco’s phosphate buffered saline (no Ca or Mg ions) (PBS) followed by centt$fugation at 150 g for 5 min. The cells were culture flasks (Falcon Labware) containing then plated in 25 cm Dulbecco’s modification of Eagle’s minimum essential medium (DMEM, Gibco Bio-Cult) with 10% (v/v) fetal calf serum (Gibco Bio-Cult) and 50 pglml gentamicin sulphate (Sigma London Chemical Co.). The flasks were maintained in a humidified atmosphere at 37’C containing 10% C02. After 16 h, non-adherent cells were removed by washing the plate 3 Experiments were performed in 5 ml of DMEM. times with 10 ml DMEM. Cells removed from the lung were pelleted by Microscopy. centrifugation at 150 g and fixed in 0.1 M cacodylate buffer pH 7.4 containing 3% (v/v) glutaraldehyde and 5 mM CaC12. The fixed cells were dehydrated in ethanol solutions of increasing sequentially
164
FEBRUARY
1984 VOL. 27 NO. 2
PROSTAGLANDINS
concentrations. They were then embedded in Epon 812 epoxy-resin (Shell Chemicals) and sectioned with a diamond knife. Plastic sections (1 urn) were stained with 1% (w/v) toluidine blue in 1% borax buffer Thin sections (60-70 nm) mounted and examined by light microscopy. saturated on 400 mesh copper grids were double stained with and an aqueous solution of 0.1% (w/v) uranylacetate in 100% methanol, lead citrate. by electron microscopy These sections were ex yined (80 Kv) at magnifications of 3.3-6.6 x 10 . Opsonized zymosan A. Lyophilized zymosan A from S. cerevisiae was obtained from Sigma London Ltd. A suspension of zymosan A (20 mg dry weight/ml) in PBS was boiled for 30 min. The particles were then washed 3 times in 10 ml PBS by resuspension and centrifugation at 3000 g for 5 min. The zymosan A particles (4 ml of 20 mglml in PBS) were opsonized with 2 ml pooled human serum by incubation at 37OC for 30 min. The opsonized zymosan A was finally washed 3 times as before with 10 ml PBS, and resuspended in PBS (20 ma/ml). Measurement of prosiaglandins and leukotriene B,, thromboxane B At the end of each timed’incubation of cells in DMEhl (5 ml), 3.5 rr?l’of DMEM was removed and added to 0.35 ml 0.5 M Tris-HCI buffer pH 7.4. Ty each sample was then a2dded 10 ng each of the folpwing standards: [ H4] prostaglandin F [ H lprostaglandin E and [ H ]6-oxo-prostaglandin F The sar@ies w&e centrifuged a3 3000 g 4or 5 min to remove \& zymosan A particles, and the supernatants frozen and stored at -2OOC. redistilled analytical grade solvents In the analysis of the samples, were used. Silica and uBondapak Cl8 reverse-phase Sep-Pak cartridges were supplied by Waters Assoc. (Inst.) Ltd. Sephadex LH-20 was obtained from Pharmacia Fine Chemicals. Methoxyamine hydrochloride (Eastman Co.) was recrystallized before use from ethanol containing about 1% (v/v) concentrated HCI. Pentafluorobenzyl (PFB) bromide (Fluorochem, UK) and bis-(trimethylsilyl)-trifluoroacetamide (Pierce Co.) were used without further purification. Prostaglandins, thromboxane B and the deuterated standards were kind gifts of the Upjohn Corn pans. Leukotriene B was3 a kind gift of Dr. J. Rokach lh,lSH-leukotriene B (30 Cilmmol) (Merck Frosst Canada Inc.). 5S,12Swas purchased from New England Nuclear, Boston, MA. dihydroxy-6,8,10,14-(E,Z,E,Z)-eicosatetraenoic 5S,l2Sacid, and 5S,l2R-dihydroxy-6,8,10,14(E,E,E,Z)-eicosatetraenoic acids were kind gifts of Dr. John Salmon. 8S,l5S-dihydroxy-5,9,11,13-(Z,E,Z,E)eicosatetraenoic and erythro-14,15-dihydroxy-5,8,10,12-(Z,Z,E,E)eicosatetraenoic acids Alan Brash and were kind gifts of Dr. Dr. John Murray. Extraction. Samples for analysis were thawed over 30 min at room temperature. The fluid was acidified to pH 3.5 with 2 M HCI and applied to a pre-washed reverse-phase C uBondapak Sep-Pak. The Sep-Pak was washed with 2 ml of distilldi water, and the eicosanoids eluted with 7 ml of ethyl acetate. This solution was applied to a straight phase silica Sep-Pak, and the ethyl acetate that eluted was discarded. The Sep-Pak was washed with 2 ml ethyl acetate and the eicosanoids eluted with 5 ml methanol. The methanol was evaporated under N2 and the residue transferred with a further 400 ul methanol to a small vial.
FEBRUARY
1984 VOL. 27 NO. 2
165
PROSTAGLANDINS
Derivatisation (a) Methoxlmation. The methanol was evaporated under N2 and with methoxyamine the dry extracts were treated 100 ul of hydrochloride in dry pyridine (5 mg/ml). The mixture was allowed to stand overnight at room temperature, and the pyridine then evaporated under N . (b)* Pentafluorobenzyl esterification. The crude residue from the methoximation step was dissolved In 36 ul acetonitrile. To this was added 10 ul 35% (v/v) PFB bromide acetonitrile and 10 ul in N ,N-diisopropylethylamine, and the solution heated to 40°C for 15 min. After cooling, the reagents were evaporated under N2, and the residue dissolved in 400 ul dichloromethane. The solution was applied to a short column of Sephadex LH-20 pre-swollen in dichloromethane and the derivatives eluted with a further 3 ml dichloromethane. (c) Trimethylsilylation. dichloromethane The solution was under N, residue evaporated the dissolved in 50 ul and his-(trimethylsilyl)-triflforoacetamide (bis-TMS-trifluoroacetamide) . After standing overnight at room temperature, the reagent was evaporated under dry N and the residue dissolved in n-dodecane ready for qas chromatoqraphic analysis. f mass spectrometric -Capillary caumn gas chromatography. Gas chromatography was carried out on a Finnigan 9600 with a glass-lined injector maintained at Injections were made in the
166
FEBRUARY
1984 VOL. 27 NO. 2
PROSTAGLANDINS
E 2 as the standard for prostaglandin leukotriey B , 12H lprostaglandin D2 and [ H f6-oxo-4prostaglandin F as the standard for thromboxane Furthir details of the prostai?andin analysis have been published B2’ previously (20.21). Measurement of N-acetyl+D-glucosaminidase 3.2.1.30) (EC zt$i;vied p;;;dmz;h;;2)employed was a modification of previously Volumes of 50 ul of the cell supernatants were incubated with 450 ul 4 mM p-nitrophenyl-N-acetyl+Dglucosaminide (Sigma, London, UK) in 40 mM citrate buffer pH 4.5 containing 5% (v/v) methanol. The incubation was allowed to proceed for 2-8 h, depending on the enzyme activity in the supernatant, and the reaction terminated by the addition of 1.5 ml 2 M glycine pH 10.7. The production of p-nitrophenol was monitored by the change in absorbance at 410 nm. The production of p-nitrophenol was linear with time and increasing enzyme concentration (to 5 mU / reaction volume) up to a final OD reading of 1.3 (1 unit of enzyme activity = 1 umol product/min). The protein content of the cultured macrophages was determined by a modification of the method of Lowry et al. (23). RESULTS The recovery of prostaglandins and thromboxane B2 through the extraction 2nd derivatization procedures was >85%. The recovery of H-leukotriene B4 carried through the extraction and 1 ng of derivatization procedure was 36% (S.D. = 5.1; n = 6). The negative ion mass spectrum (Fig. 1) of the bis-trimethylsilyl ether pentafluorobenzyl ester derivative of leukotriene Ba4 showed an intense ion at m/z -479 corresponding to [M-PFB] . This ion carried 20% of the tot2 Ton cur_rent. Fragment ions were also obse_rved at m/z -389 [M-PFB-TMSOH] and m/z -299 [M-PFB-(TMSOH) ] . The-i&s at -m/z -479 and m/z -389 were used for quanta 3 ative selected ion monitoring of lelkotriene B4. The limit of sensitivity was 2 pg on column. The retenjion time for leukotriene B was 7.31 min. and the retention time for [ H4lprostaglandin F20 was ‘4 .44 min under identical chromatographic conditrons. Quantification of leukotriene B was carried out by interpolation from linear regression curves of %uthentic standards in the range 0.1-10 ng. These were constructed by measurement of peak areas of the (M-PFB)ion at m/z -479 and the (M-PFB-TMSOH)ion at m/z -389 from leukotriene B -aEd determigng the ratio of these ionsthe (M-PFB)ion at -m?z -573 from ( H )-PGF . The area ratios were expressed as a percentage and a linelr reg&sion of log (concentration of leukotriene B in ng) against log (area ratio %) carried out. The equation of the tegression line for m/z -479 was y = exp (0.751 + 0.989 log x) r = 0.994. The equation ;f-the regression line for mlz -389 was y = exp (0.703 + 1.046 log x) r = 0.992. The Kovats Fecntion indices (24) were determined for a number of isomeric dihydroxy eicosatetraenoic acids using PFB-esters of saturated fatty acids as standards. The values obtained (Table 1) indicate that leukotriene B can be separated from these compounds on Sil-5 fused silica capillar 4 columns. Leukotriene B was baseline from separated to 5S,12S-dihydroxy-6,8,10,14-(r,Z,E,Z)-eicosatetraenoic acid, a selected
FEBRUARY
1984 VOL. 27 NO. 2
167
PROSTAGLANDINS
299
106
150
200
300
250
._.A
1
389
M-PFB -
ion chemical PFB-ester,
Table I. The Kovats eicosatetraenoic acids. Fatty
retention
ionisation mass spectrum ether derivative. -bis-TMS
indices
for
Acid
selected
Kovats
of
dihydroxy-
Retention
Index erythro-14,15-dihydroxy-5,8,10,12-(Z,Z,E,E)eicosatetraenoic 5S,12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-eicosatetraenoic 5S,12R-dihydroxy-6,8,10,14-(Z,E,E,Z)-eicosatetraenoic 8S,lSS-dihydroxy-5,9,11,13-(Z,E,Z,E)-eicosatetraenoic 5S,12S-dihydroxy-6,8,10,14-(E,E,E,Z)-eicosatetraenoic 5S,12R-dihydroxy-6,8,10,14-(E.E.E,Z)-eicosatetraenoic
168
2222 2229 2302 2312 2455 2455
FEBRUARY
1984 VOL. 27 NO. 2
PROSTAGLANDINS
ion current profile of the channel used to monitor m/z -479 is shown in Figure 2b. the ionization Under conditions - employed, little fragmentation of the 5S,lZS-dihydroxy fatty acid occurred. Thus, monitoring the mlz -389 ion a response could only be observed for leukotriene B (Figure 2a). The 5S.12S and 5S,lZR-dihydroxy6,8,10,14-(E,e,E,Z)-eicosatetraenoic acids co-eluted (Kovat’s indices 2455), however they were well resolved from leukotriene B4 in both the m/z -389 (Figure 2c) and m/z -479 channels (Figure 2d). -Examination by light>?roscopy of the cells obtained from human airways revealed that >95% of the cells present were a single morphological type. These were shown to be macrophages by electron microscopic examination of their ultrastructure. On a few occasions, the cells were heavily contaminated with desquamated epithelial cells, and on these occasions there was little or no adhesion of the macrophages to the culture flasks, These specimens were always discarded. Those specimens found suitable for these studies (about 4 out of every 5 samples) were plated as described in the Methods section. The plating efficiency varied between 55% and 90%, gnd the numbers gf adherent cells/flask varied between 0.5 x 10 and 2 x 10 . Microscopic examination of the adherent cells revealed that >98% were Adherent cells were examined for Trypan blue exclusion macrophages. before and after 5 h incubations in the absence or presence of zymosan A opsonized with human serum. The results showed that viability of the cells, as measured by this technique, was >90%. The non-adherent cells were mostly erythrocytes and lymphocytes. Cultured macrophages in replicate flasks were incubated for up to 5 h in the absence or presence of 1.2 mglml zymosan A opsonized with human serum. The release of prostaglandins D2, E2, F20, and into the culture medium in a typical experiment are thromboxane B shown in Fig 23 .A and B. A wide variation was observed in the concentrations of these eicosanoids, and no 6-oxo-prostaglandin Fla, the stable hydrolysis product of prostacyclin, was detected in any of the experiments. The release of all eicosanoids examined, with the exception of 6-oxo-prostaglandin Fla, was facilitated by opsonized zymosan A particles, although there was a significant release in the absence of zymosan A. The levels of prostaglandins released from macrophages were remarkably consistent from lung to lung, with the exception of prostaglandin D2. The results of eicosanoid release from cells obtained at 4 separate bronchoscopies were as follows (5 h after the addition of opsonized zymosan): prostaglandin E = 2.73 2 0.45 nglmg cell protein,prostaglandin F2 = 7.08 2 2.70 ng?mg cell protein, thromboxane B2 = 65.2 2 14.8 ng?mg cell protein and prostaglandin D = 24.5 + 37.4 nglmg cell protein (prostaglandin D2 range = 3.05 to Sd.4 nglmg cell protein; results show means + S. D. ) . In other experiments (data not presented), the release of these prostaglandins from alveolar macrophages was measured for times up to There was a gradual increase in prostaglandin and thromboxane 8 h. B2 concentration in the culture medium, with little tendency to plateau even at 8 h. In the experiment presented in Fig 3D, the culture medium was also analysed for its content of the lysosomal enzyme N-acetyl+D-glucosaminidase. The release of the enzyme from alveolar
FEBRUARY
1984 VOL. 27 NO. 2
169
PROSTAGLANDINS
macrophages opsonized unstimulated
was thus monitored The zymosan A. cells was increased
100- A
for 5 h in the absence or presence gradual release of the enzyme by the addition of zymosan A.
of in
- c
-
D
+=+Qo 1-
180
Scans Elution profiles of dihydroxyeicosatetraenoic acids Fig. 2. Results show from a capillary column gas chromatograph. (retention time = 7.31 the separation of mixtures of LTB SS,!‘tl2S-dihydroxy-6,8,10,14and min) (E,Z,E,Z)-eicosatetraenoic acid (retention time = 5.25 min) in 5S,12S and separation of A and the and B, 5S,12R-dihydroxy-6,8,10,14(E,E,E,Z)-eicosatetranoic acids (retention times both = 8.69 min) from LTB in C and D. These compounds were identified by selected ic?n monitoring at m/z -389 (A,C) or -m/z -479 (B,D). --
170
FEBRUARY
1984 VOL. 27 NO. 2
PROSTAGLANDINS
their capacity to also examined for release Cells were 3C) reveal a time course for the The results (Fig. leukotriene 6 . differs significantly from that observed BQ that release of le$kotriene prostaglandins, thromboxane and release of the for the B2 There was little or no release of N-acetyl+-D-glucosaminidase. leukotriene BG from cells cultured for 5 h in the absence of opsonized addition of zymosan increased the release of However, zymosan A. This result compares with the leukotriene B4 at least 20-fold at 2 h. 2-fold change after zymosan observed in the release of the
Hours D2 ( A A ), E2 B ( + * 1, are ’in F2% of2 N-acetyl-P-Dshown glucosaminidase ( + 0 )‘, is :~~wnr%a~e and the release of leukotriene B ( l 0 ) in C. Human alveolar macrophages the times shown in the absence (open were culture d for symbols) or presence (closed symbols) of 1.2 mg/ml opsonised zymosan A. Results are means (+ S.E.M.) of the concentrations of these compounds in the culture medium of The units in A, B, C are expressed as triplicate flasks. and in D the units are “enzyme activity” ng/mg cell protein, (nmol product/h) released per mg cell protein.
Wi
FEBRUARY
The
release ( n 0 and
of
), B
1984 VOL. 27 NO. 2
prostaglandins and thromboxane
171
PROSTAGLANDINS
Table -of human
2.
The effect of leukotriene B alveolar macorphag&.
sodium flurbiprofen thromboxane B2
Concentration Eicosanoid
Prostaglandin D2 Prostaglandin E2 Prostaglandin F 6-oxo-prostagla&in Thromboxane B2 Leukotriene B4
Control
Fla
on the synthesis and prostaglandins
(ng/mg
cell
and from
protein)
Flurbiprofen
4.88 3.01 3.18 <1.3 30.7 5.77
<1.3 <1.3 <1.3 <1.3 2.09 10.8
Alveolar macrophages were cultured and the release of eicosanoids in measured as described Methods. Flurbiprofen (1 uM) was added to the cells 2 min before the addition of zymosan A. The cell supernatants were harvested 3 h after the addition of opsonized zymosan A The results are the means of duplicate culture particles. flasks containing cells from one patient. prostaglandins, thromboxane B and the enzyme. Furthermore, no additional accumulation of leuko 2t riene B was observed in the culture medium after 2 h for times up to 8 h (da?a not presented). There was a significant variation in the levels of leukotriene B4 released from cells from 4 different patients (range = 1.15 to 15.5 nglmg cell protein) with a mean (2 S.D.) of 7.28 + 5.9? nglmg cell protein. The effect of sodium flurbiprofen on the synthesis and release of prostaglandins and thromboxane B2 was measured. A solution of 1 uM sodium flurbiprofen inhibited significantly synthesis of the prostaglandins D2, E2, F20 and thromboxane B2 (Table 2) in the presence of 1.2 mg/ml opsonized zymosan A. The synthesis and release of leukotriene B however, was increased 2-fold. The inhibition by sodium flurbiprofen in the of thromboxane B2 synt ‘h’esis and release was selected presence of opsonized zymosan A compared at concentrations of sodium flurbiprofen (Fig. 4). The results reveal a concentration for 50% inhibition by sodium flurbiprofen of 30 nM. In other experiments (data not presented), the effect of sodium flurbiprofen (100 uM) was examined on the release of N-acetyl-BD-glucosaminidase in the absence or presence of 1.2 mglml of opsonized zymosan A. There was, however, no effect of sodium flurbiprofen on basal or zymosan-stimulated enzyme release over a period of 5 h. DISCUSSION Previous methods for the analysis of endogenous leukotriene B4 have relied on the use of radioimmunoassay (19) or high performance liquid chromatography (25). We have examined the use of gas chromatography/mass spectrometry for the quantitative analysis of leukotriene B4. The technique should provide a method with a degree
172
FEBRUARY
1984 VOL. 27 NO. 2
PROSTAGLANDINS
[Na flurbiprofen]
M
4. The effect of sodium flurbiprofen on the *sis and release of thromboxane 62. The results were obtained by analysis of cell supernatants (duplicate flasks at each concentration) harvested 3 h after the addition of opsonized zymosan A particles. Cells were cultured in the absence or presence of flurbiprofen at the concentrations shown, and in the experiment, flurbiprofen was added to the cells 2 min before the addition of zymosan A.
of specificity that cannot be attained with either high performance liquid chromatography or radioimmunoassay. Electron impact mass spectrometry causes extensive fragmentation of the leukotriene molecule (26) so that selected ion monitoring techniques required for gas chromatography-mass relatively spectrometric analysis would be insensitive. However, it has been reported recently that negative ion chemical ionization mass spectrometry can be used for the efficient ionization of prostanoids (27). The technique relies on thermal electron capture in the gas phase, and the process is facilitated by prior conversion of the prostanoid carboxyl group to the PFB ester. Paradoxically, once the electron capture has taken place, the PFB moiety is lost and the stabilized carboxylate anion is formed. These
FEBRUARY
1984 VOL. 27 NO. 2
173
PROSTAGLANDINS
[M-PFB]ions are ideal for specific and sensitive quantitative analysis which suggested that the same technique of prostaglandins (20,21), could be used for the analysis of leukotriene B . was converted to its PBk ester bis-trimethylsilyl Leukotriene B ionization and subjec t ed to negative ion chemical mass ether It had similar characteristics to spectrometric analysis. the prostanoids although it showed a greater tendency to fragment Selected ion monitoring analysis of the [M-PFB] ion (Fig. 1). indicated that the limit of sensitivity for leukotriene B4 was 2 pg on A quantitative assay was designed in yhlch two ions, column. [M-PFE]and [M-PBF-TMSOH] , were monitored and [ H4lprostaglandin as the internal standard for quantitative analysis. F 2a. was employed The analysis chromatography/mass of leukotriene B4 by gas spectrometry is complicated by the possibility of interference from an isomeric dihydroxyeicosatetraenoic acid. If the specific hydrolase became enzyme which converts leukotriene A to leukotriene B saturated then aqueous hydrolysis wou l4d result in the for!nation of 5S,12S and 5S,12R-dihydroxy-C,8,10,14-(E,E,E,Z)-eicosatetraenoic acids The retention data in the literature suggests that these two (28). compounds (C values 24.8 as methyl esters) (29) would separate from There are no leukotriene B4 (C value 23.6 as methyl ester) (29,30). reported retention times for the corresponding PFB-esters so these were determined, and the Kovats Indices (24) calculated by interpolation from appropriate PFB-esters of saturated fatty acid standards (Table 1). no possibility of These data clearly established that there was interference in the assay from the aqueous hydrolysis products of The other possible interference was from the double leukotriene A . Iipoxygenase %roduct of arachidonic acid, 5S,12S-dihydroxy-6,8,10,14(E,Z,E,Z)-eicosatetraenoic acid (30.31). The retention data in the literature on this compound is equivocal. Three studies report C values on the methyl ester of 22.4 (1% SE-30) (31), 23.6 (1% SE-30) (30) and 23.7 (1% SP 2100) (32) respectively. It was noted in one report that the gas chromatography properties of the compound were poor (32). A similar observation was made previously on mono-hydroxy fatty acids in which there was a similar E,Z geometry at Perhaps the poor chromatographic C-6 and C-8 respectively (33). characteristics of the compound account for the discrepancy in the retention data. The PFB-ester also appeared to have poor characteristics although baseline resolution could be obtained between the double lipoxygenase B4 (Figure 2b). In addition, the product and leukotriene Under the fragmentation pattern of the molecule was quite different. -389 there ion at ionization conditions was no employed m/z to (M-PFB-TMSOH)(Figure 2a). This -iOn was corresponding employed for quantitative selected ion monitoring analysis and so there could be no possibility of interference in the assay for leukotriene B4 acid. 5S,12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-eicosatetraenoic by Finally, the Kovats indices were determined for two other isomeric 8S,15S-dihydroxy-5,9,11,13-(Z,E,Z,E)-eicosatetraenoic and acids, erythro-14,15-dihydroxy-5,8,10,12-(Z,Z,E,E)-eicosatetraenoic acids to show that they could not interfere in the assay (Table 1). the experimental design was similar in In the handling of cells, By maintenance many respects to methods described previously (10).
174
FEBRUARY
1984 VOL. 27 NO. 2
PROSTAGLANDINS
of the cells in monolayer culture, non-viable macrophages, erythrocytes and lymphocytes were removed from the culture flask as non-adherent cells in the washings. The time courses for the release of prostaglandins D E2, F and the lysosomal enzyme N-acetyl+-Dglucosaminidase *were SI‘I#?I ar, with a gradual increase in their concentrations in the culture medium, and little tendency to plateau over 8 h. In this respect these results are similar to those obtained with mouse peritoneal (10) and rabbit alveolar macrophages (11,12). The release of lysosomal enzymes accompanies phagocytosis, and is probably initiated by the fusion of the internalized ‘or pinocytic vesicle with a lysosome. Whether any benefit to the host accompanies the in vivo release of lysosomal hydrolases from macrophages into tt% extracellular environment is uncertain. The finding that inhibition of the cyclooxygenase activity (Table 2) with flurbiprofen had no effect on the release of the acid hydrolase N-acetyl+-D-glucosaminidase has been observed in similar experiments with animal macrophages (10,ll). There is an extensive literature on the different prostaglandins and their relative proportions released from macrophages under resting conditions or after stimulation (1 O-l 6.34). There is some consistency in the results, but differences have been observed between species and even among macrophages from different anatomical sites of the same species (34). The relative concentrations of arachidonic acid metabolites released from macrophages are altered only slightly in stimulated cells when compared to those in a resting state (34). (7,8,10-14,34), F Significant concentrations of prostaglandins E (ll-14,34), D2 (ll-14), C-0x0-Fl (8,10-14,16,3& and thromboxane 6” (16,34) are released from macro&ages of many species, and in moss reports prostaglandins E2, C-0x0-Flu and thromboxane B2 are the most abundant. Early reports of significant synthesis and release of prostaglandins El and F,o are not now generally accepted, and are probably the consequence of problems with analysis not appreciated at the time. In the present communication data are presented for the release of prostaglandins generated from endogenous substrate, thus permitting quantitative measurement of the total prostaglandins released in vitro Inspection of the results reveals from human alveolar macrophages. was the most abundant metabolite of arachidonic that thromboxane B acid, although sign1 .3 icant, but variable, levels of prostaglandin D2 were also observed. Prostaglandins E2 and F were also released at lower concentrations, but no C-oxo-prostaglan C!” In F was detected in the The absence of I%-oxo-prostaglandin F supernatants of these cells. in human alveolar macrophages is similar to that observed in alveoli? macrophages of mice (34), and it is intriguing to note that in the same lungs report, mouse pulmonary macrophages obtained from chopped released (rather than by airway lavage) synthesised and 6-oxo-prostaglandin F in response to zymosan. Human alveola r’” are known to macrophages synthesise leukotriene B4 (S), and in the present report quantitative measurement of its release in vitro has been provided for the first time. In based on capillary column gas addition, its physico-chemical properties, chromatography and negative ion chemical ionization mass spectrometry have identified this product with great certainty. The original report (5) identified leukotriene B4 on the basis of its chromatographic and UV
FEBRUARY
1984 VOL. 27 NO. 2
175
PROSTAGLANDINS
spectral properties. In addition, a bio-assay employing the neutrophil polymorphonuclear leucocyte chemotactic response demonstrated similar biological activities to authentic leukotriene B4. There was little or no release of leukotriene B4 from resting macrophages and addition of opsonized zymosan A particles resulted in an apparent more rapid release of leukotriene B than the cyclooxygenase products, with no additional release after tlhe first 2 h. However, the possibility that leukotriene B is metabolised in this system cannot be discounted. There w$s little or no synthesis of thromboxane B , or any of the prostaglandins measured, in the presence of 1 uM sod&m flurbiprofen, a potent inhibitor of cyclooxygenase activity Both the (35). cyclooxygenase and I ipoxygenase enzymatic pathways represent minor metabolite pathways for clearance of free arachidonic acid, the major portion being re-esterified and incorporated into membrane lipid . Nevertheless, inhibition of with 1 uM cyclooxygenase sodium flurbiprofen (Table 2) increased nearly 2-fold the synthesis and release of the lipoxygenase product leukotriene B4. The significance of this observation in relation to the regulation of lipoxygenase activity remains obscure, but may simply reflect an increase in substrate availability. In conclusion, the results provide quantitative measurement of leukotriene B thromboxane B and seve ra I prostaglandins from endogenous su b strate of human a 12 veolar macrophages cultured in vitro. The analyses were based on capillary column chromatography coupledo negative ion chemical ionization mass spectrometry. ACKNOWLEDGEMENTS Financial support Research Council (UK)
from the Wellcome Trust is gratefully acknowledged.
(UK)
and
the
Medical
REFERENCES
1.
Borgeat, P., and B. Samuelsson. acid by rabbit polymorphonuclear 1979. -254: 2643,
3.
Cunningham, F.M., H. R. Carter, M.J.H. Smith, A.W. Ford-Hutchinson and M.A. Bray. Aggregation of polymorphonuclear leucocytes (PMNs) by leukotriene B : Effects of cyclooxygenase products and metabolic inhibitors. A!gents and Actions 583, -11: 1981.
4.
Smith, M.J.H., Leukotriene B Pharmacol. -32:
176
A.W. a potential 517, 1980.
Fels, A.O.S., N.A. Z.A. Cohn, and W.A. leukotriene B4. Proc.
activities
of
leukotriene
of arachidonic Biol. Chem.
Smith, Actions
:
Biological 1981.
J.
2.
5.
M.J.H. -11: 571,
Transformation leukocytes.
Ford-Hutchinson, mediator of
B4.
and inflammation.
Agents
M.A. J.
and
Bray. Pharm.
Pawlowski, E.B. Cramer, T.K.C. King, Smith. Human alveolar macrophages produce Natl. Acad. Sci. USA -79: 7866, 1982.
FEBRUARY
1984 VOL. 27 NO. 2
PROSTAGLANDINS
6.
Rouzer, C.A., W.A. Scott, Z.A. Cohn, P. Blackburn, and J.M. Manning. Mouse peritoneal macrophages release leukotriene C in response to phagocytic stimulus. Proc. Natl. Acad. Sci. USA -77: 4928, 1980.
7.
Rouzer, C.A., Prostaglandin receptor-ligand 1980.
8.
Rouzer, C.A., W.A. Scott, A.L. Hamill, F-J. Liu, D.H. Katz, Z.A. Cohn. Secretion of leukotriene C and other arachidonic metabolites by macrophages challenged with immunoglobulin immune complexes. J. Exp. Med. -156: 1077, 1982.
9.
Rankin, Brashler, C4 from
10.
Bonney, R.J., P.D. Wightman, P. Davies, S.J. Sadowski F.A. Kuehl, and J.L. Humes. Regulation of prostaglandin synthesis and of the selective release of lysosmal hydrolases by mouse peritoneal macrophages. Biochem. J. -176: 433, 1978.
11.
Hsueh, W., prostaglandin phagocytosis 1979.
12.
Hsueh, W., F. Gonzalez-Crussi, synthesis in different phases of Nature -283: 80, 1980.
13.
Hsueh, W., U. Desai, F. Gonzalez-Crussi, R. Lamb, and A. Two phospholipase pools prostaglandin for synthesis macrophages. Nature 1980. -290: 710,
14.
Hsueh, W., R. Lamb, and F. Gonzalez-Crussi. phospholipase A2 activity and prostaglandin biosynthesis calmette-guerin-activated alveolar macrophages. Biochim. Acta. 1982. -710: 406,
15.
Stenson, W.F., and C.W. and immunity. J. Immunol.
16.
Cook, J.A., prostacyclin macrophages.
17.
Flower, R.J., phospholipase-A2 1976. -25: 285,
FEBRUARY
W.A. synthesis interaction.
Scott, .I. Kempe, by macrophages Proc Natl. Acad.
J.A., M. Hitchcock, and P.W. Askenase. alveolar macrophages.
C. Kuhn, secretion and lysosomal
and Z.A. Cohn. requires a specific Sci. USA -77: 4279,
W. Merrill, M.K. Bach, J.R. IgE-dependent release of leukotriene Nature -297: 329, 1982.
and P. Needleman. Relationship alveolar by rabbit macrophages enzyme release. Biochem. J. -184:
and E. Hanneman. phagocytosis in lung
Parker. -125: 1,
Prostaglandins, 1980.
1984 VOL. 27 NO. 2
of to 345,
Prostaglandin macrophages.
Chu. in
Decreased in bacillus Biophys.
macrophages,
W.C. Wise, and P.V. Halushka. Thromboxane produced by lipopolysaccharide-stimulated J. Reticuloendothel. Sot. -30: 445, 1981. and G.J. in prostaglandin
and acid E
A and pet-1.?oneal
Blackwell. The importance of biosynthesis. Biochem. Pharmac.
177
PROSTAGLANDINS
18.
Bell, R.L., D.A. Kennerly, N. Stanford, and P.W. Majerus. a pathway for arachidonate release from human Diglyceride lipas?: platelets. Proc. Natl. Acad. Sci. USA -76: 3238, 1979.
19.
Salmon, J.A., radioimmunoassay
P.M. Simmons, for leukotriene B4.
K.A., J. Wellby, gas chromatography Mass Spectrom. -10:
and R.M.J. Prostaglandins
and I.A. Blair. mass spectrometry 83, 1983.
-24:
20.
Waddell, column Biomed.
21.
Waddell, K.A., C. Robinson, M.A. Orchard, Blair. Quantitative analysis C.T. Dollery, and I.A. acid metabolites in biological fluids using GCINICIMS. Spectrom. Ion Phys. 1983. -48: 233,
22.
Schwartz, L.B., K.F. Austen, and S.I. Wasserman. release of p-hexosaminidase and 8-glucaronidase from serosal mast cells. J. Immunol. 1979. -123: 1445,
23.
Lowry, O.H., N.J. Protein measurement 1951. -193: 265,
24.
Wehrli, A., charakterisierung 2709, 1959.
and organic
25.
Mathews, W.R., leukotrienes by Biochem. 96, -lib:
J. Rokach, high-pressure 1981.
26.
Ford-Hutchinson, A.W., M.A. Bray, Leukotriene B, and M.J.H. Smith. aggregating substance released leukocytes. Nature -286: 264, 1980.
Rosebrough, with the folin
E.
28.
Maycock, A.L., Jr. Leukotriene cell-free system 1982.
29.
and B. Samuelsson. Borgeat, P., polymorphonuclear leukocytes. hydroxylated compounds. J. Biol.
178
S.E. Barrow, Prostacyclin is 23: 579, 1982. -
and
Blair, I.A., C.T. Dollery. Prostaglandins
Barrow, S.E. of arachidonic Int. J. Mass
Immunologic purified rat
Gas-chromatographische Helv. Chim. Acta
R.C. liquid
K.A. not a
M.S. Anderson, Preparation A4: to leukotriene
Combined capillary of prostanoids.
A.L. Farr, and P.J. Randall. phenol reagent. J. Biol. Chem.
Kovats. verbindungen.
27.
Palmer. A 225, 1982.
a
-42:
Murphy. Analysis of Anal. chromatography.
M.V. Doig, M. E. Shipley, potent chemokinetic and polymorphonuclear from
Waddell, circulating
P.J. Lewis, hormone in
and man.
D.M. De Sousa, and F.A. and enzymatic conversion B4. J. Biol. them. -257:
Kuehl, in a 13911,
Metabolism of arachidonic Structural analysis of Chem. 1979. -254: 7865,
acid in novel
FEBRUARY
1984 VOL. 27 NO. 2
PROSTAGLANDINS
P.,
S. of arachidonic
Transformation and
Med.
557,
P. acid
Vallerand, and in leukocytes.
P. Isolation
and
1981.
G.
31.
Lindgren, .A., novel hydroxylated polymorphonuclear
32.
Maas, R.L., A.R. Brash. the lo-carbon A,, by human
33.
Boeynaems, J.M., and W.C. Hubbard. Preparation, purification, characterization and assay of hydroxyand hydroperoxyeicosatetraenoic acids. In: Prostaglandins, prostacyclin and thromboxane measurement. Boeynaems, J.M. and Eds Herman, A.G., Nijhoff, The Hague, p. 167, 1980.
34.
Rouzer, Synthesis by mouse
35.
Cockbill, S.R., the abilities of to inhibit the human blood
Editor: Received: Accepted:
FEBRUARY
Hansson, and B. Samuelsson. Formation of eicosatetraenoic acids in preparations of human leukocytes. FEBS Lett. -128: 329, 1981.
C.D. Ingram, D.F. Taber, J.A. Oates, and Stereospecific removal of the D hydrogen atom at of arachidonic acid in the biosyn Fzhesis of leukotriene leukocytes. J. Biol. Chem. 1982. -257: 13515,
C.A., W.A. Scott, of leukotriene C and pulmonary macrophages.
Elisabeth 6-22-33 12-20-83
A.L. other
Hamill, and Z.A. Cohn. arachidonic acid metabolites J . Exp. Med. -155: 720, 1982.
S Heptinstall, and P.M. Taylor. A comparison of flurbiprofen and indomethacin acetylsalicylic acid, release reaction and prostaglandin synthesis in Pharmac. 73, 1979. platelets. Br. J. -67:
Granstrom
1984 VOL. 27 NO. 2
179