VOLUME NUMBER
Hyper-IgE
88 5
18. Sampson HA, Buckley RH. Human IgE synthesis in vitro: reassessment.J Immunol 1981;127:829-34. 19. Gleich GJ, Averbeck AK, Svedlund HA. Measurement of IgE in normal and allergic serum by radioimmunoassay. J Lab Clin Med 1971;77:690-8. 20. Vercelli D, Jabara HH, Cunningham-Rundles C, et al. Regulation of immunoglobulin (1g)E synthesis in the hyper-IgE syndrome. J Clin Invest 1990;85:1666-71. 21. King CL, Gallin JI, Malech HL, Abramson SL, Nutman TB. Regulation of immunoglobulin production in hyperimmunoglobulin-E recurrent-infection syndrome by interferon-y. Proc Nat1 Acad Sci USA 1989;86: 10085-g. 22. Del l%:te G, Tiri A, Maggi E, et al. Defective in vitro production of y-interferon and tumor necrosis factor by circulating T cells from patients with the hyperimmunoglobulin E syndrome. J Clin Invest 1989;84:1830-5. 23. Finkelman FD, Holmes J, Urban JF Jr, Paul WE, Katona IM. T help requirements for the generation of an in vivo IgE response a late acting form of T cell help other than IL-4 is required for IgE but not for IgGl production. J Immunol 1989;1,12:403-8.
B cells
are refractory
to IL-4
24. Vercelli D, Jabara HH, Arai K-I, Geha RS. Induction of human IgE synthesis requires interleukin-4 and T/B cell interactions involving the T cell receptor/CD3 complex and MHC class II antigens. J Exp Med 169: 1295-1307. 25. Li JTC, Yunginger JW, Reed CE, Jaffe HS, Nelson DR, Gleich GJ. Lack of suppression of IgE production by recombinant interferon gamma: a controlled trial in patients with allergic rhinitis. J ALLERGYCLIN IMMUNOL 1990;85:934-40. 26. Boguniewicz M, Jaffe HS, Izu A, et al. Recombinant gamma interferon in treatment of patients with atopic dermatitis and elevated IgE levels. Am J Med 1990;88:365-70. 27. Gamkrelidze A, Bjorksten B. The determination of IgE synthesis in vitro: methodological aspects. J Immunol Methods 1990;130:9-13. 28. Massicot JG, Ishizaka K. Workshop on measurement of in vitro IgE synthesisand regulation of IgE synthesis.J ALLERGY CLIN IMMUNOL 1986;77:544-54. 29. King CL, Poindexter RW, Ragunathan J, et al. Frequency analysis of IgE-secreting B-lymphocytes in persons with normal or elevated serum IgE levels. J Immunol 1991;146:147883.
Lysophosphatidylcholine induces mast cell secretion and protein kinase C activation Diana L. Marquardt,
MD, and Linda L. Walker, BS San Diego, Calif.
Lysophosphatidylcholine (lyso-PC), a natural product of phospholipase A, activity, induced the secretion of both granule-associated @hexosaminidase and newly generated leukotriene C, from mouse bone marrow-derived mast cells. Micromolar concentrations of lyso-PC potentiated the release of /3-hexosaminidase induced by specific antigen but not the calcium ionophore, A23187. Exogenous adenosine was relatively ineffective in enhancing P-hexosaminidase release from cells challenged with lyso PC. Lyso-PC caused a marked increase in intracellular free-calcium levels and induced the activation of protein kinase C (PKC). These effects could not be abrogated by a prolonged preincubation with pertussis toxin. Staurosporine. an inhibitor of PKC, partially inhibited the abilities of antigen and A23187 to induce P-hexosaminidase release but was ineffective when lyso-PC was the secretagogue. Lyso-PC appears to activate mast cell PKC, but its ability to stimulate mast cell mediator release appears to be related to its abiliv to elevate intracellular free calcium concentrations. (J ALLERGY CLIN IMMUNOL 1991;88:721-30.) Key words: Mast cells, protein kinase C. staurosporine, lysophosphatidylcholine
From the Department of Medicine, University of California-San Diego School of Medicine, San Diego, Calif. Supported by National Institutes of Health Grants AI-17268 and AI-25507, and the Hartford Foundation. Received for publication May 31, 1990. Revised June 3, 1991. Accepted for publication June 6, 1991. Reprint requests: Diana Marquardt, MD, c/o UCSD Medical Center, 8417, 225 Dickinson St., San Diego, CA 92103. l/1/31505
pertussis toxin, calcium,
Investigation of the biochemical steps necessary to support the release of mediators from mouse bone marrow-derived mast cells has led to the conclusion that the interactions between various receptors, G proteins, phospholipases , and protein kinases are all parts of this complex signal transduction system. At least three phospholipases have been studied extensively in various secretory cells and tissues. Phospholipase D induces the release of phosphatidic acid from phos721
722
Marquardt
and Walker
Abbreviations
Lyso-PC: DNP: LDH: BSA: BAPTA: Ag: LT: EGTA: j2P: PMA: ATP: PKC:
used
Lysophosphatidylcholine Dinitrophenyl Lactic dehydrogenase Bovine serumalbumin Bis-(o-aminophenoxy-)-ethaneN,N,N’,N’,-tetraacetic acid Antigen Leukotriene Ethyleneglycol-bis-(B-aminoethylether)N,N,N’,N’,-tetraaceticacid Radioactiveisotopeof phosphorus Phorbolmyristateacetate Adenosinetriphosphate ProteinkinaseC
pholipids, and the action of phosphatidic acid phosphohydrolase on phosphatidic acid may contribute to the production of diacylglycerol, a membrane fusogen and transient natural activator of PKC. ’ Phospholipase C activation may induce the production of diacylglycerol and inositol trisphosphates.’ Phospholipase A2 activation contributes to the production of free arachidonic acid, which may be metabolized to leukotrienes or prostaglandins, and lyso-PC, a potent membrane fusogen.3 Lyso-PC also has been reported to activate PKC over a narrow concentration range in selected cells and tissues.4. 5 Adenosine has been a modulator of mast cell secretion of particular interest because it appears to potentiate the release of mast cell mediators through the stimulation of phospholipase C activity,6 resulting in the production of natural PKC activators.’ Adenosine augments the ongoing release of mast cell granule constituents, such as histamine or P-hexosaminidase, but fails to induce mediator release on its own and fails to augment the production of LTC,.’ Therefore, it is one of the relatively small number of agents that have been reported to affect preformed mediator release but not new mediator generation. Because agents that alter PKC activity have been demonstrated to alter the ability of mast cells to respond to the mediator release enhancing effects of adenosine9 and because lyso-PC is a natural product of phospholipase A2 activation during mast cell degranulation, an investigation of the ability of possible physiologic concentrations of lyso-PC to modulate mast cell adenosine responsiveness, activate PKC , and alter the mast cell secretory process was undertaken.
J. ALLERGY
MATERIAL Cell-culture
CLIN. IMMUNOL. NOVEMBER 1991
AND METHODS conditions
Bone marrowfrom femursof BALB/c mice (Simonsen Laboratories,Gilroy, Calif.) wasculturedin 20%WEHIsupematantas a sourceof interleukin-3” and RPM1containing 10%fetal calf serum,0.1 mmol/L of nonessential aminoacids,50 pmol/L of 2-mercaptoethanol, 2 mmol/L of L-glutamine,100pg/ml of streptomycin,and 100U/ml of penicillin. With weekly cell passage, by 21 daysof culture, cellswere>90% viable puremastcells.
Assays The releaseof B-hexosaminidase from stimulatedmast cells was assessed as previously describedin detail.” Briefly, cells werepassivelysensitizedwith anti-DNPIgE (1 Kg/ lo6of cells)andchallenged for 10minuteswith DNPBSA Ag (200 ng13 x 10’ cells), or 1 pg/ml of A23187, and the B-hexosaminidase presentin the resultingsupernatantsandcell pelletswasquantitatedby the hydrolysisof P-nitrophenyl-B-o-glucosaminide substrate.L’ Mast cell supematant LTC, concentrations wereassessed in experiments similarto thoseexperiments above, followed by the useof a [‘H]LTC, RIA kit. Somecross-reactivity betweenLTC,, LTD,, and LTE, hasbeendescribedin this RIA, but mousebone marrow-derived mast cells primarily synthesizeLTC, by lipoxygenationof arachidonic acid.I2 PKC activity wasmeasured by the phosphorylation of a syntheticpeptidefrom myelin basicproteinthat can act as a specificsubstratefor PKC.” Mast cells(2 X 106/250~1) were challengedwith appropriateagentsfor 5 minutesat 37” C, and reactionswere terminatedby the additionof two equal volumesof cold buffer (40 mmol/L of Tris, 1 mmol/L of ethylenediaminetetraacetic acid, 1 mmol/L of EGTA, and 50 kg/ml eachof aprotininand leupeptin, pH 7.5), sonicationfor 10 seconds,and centrifugationat 100,000g for 30minutesat 4” C. Supematants weresaved, and pellets were resuspended in 0.5 ml of buffer (20 mm01 /L of Tris, 0.5 mm01 IL of ethylenediaminetetraacetic acid, 0.5 mmol/L of EGTA, 0.5% Triton X-100, and 25 pg/ml eachof aprotininandleupeptin,pH 7.5) andsonicated again for 10 seconds.Aliquots of supematantand pelletfractions(5 pl) wereassayed by preincubationfor 20 minutesat roomtemperaturein the absence or presenceof a pseudosubstrate inhibitor, the additionof 32PgammaATP and substrateat 30” C for 5 minutes,andspottingsamples on phosphocellulose disksand determiningthe 3zPcounts in the absenceandpresenceof the inhibitor. Intracellularfree-calciumlevelswerequantitatedby loading cellswith the fura 2 probeandmeasuringfura 2 fluorescence,aspreviouslydescribedfor this mastcell population.‘, I4 LDH activitiesin cell supematants, pellets,andsonicated pelletswereassessed with a calorimetrickit purchased from SigmaChemicalCo., St. Louis, MO. The productionof the inositolphosphates, IP, IPI, and IP,, wasdeterminedby incubatingcellsfor 18 hourswith
VOLUME NUMBER
Lysophosphatidylcholine
88 5
0 Spontaneous IZI + Antigen gzfds + Ag &Ado
and
mast
ceils
723
* *
* 1
FIG. 1. Effect of lyso-PC on Ag-stimulated mast cell P-hexosaminidase release. Cells were incubated for 20 minutes with 0 to 10 pmol/L of lyso-PC and challenged with buffer (U), DNP) for 10 minutes. Depicted BSA Ag (200 ng) (tZZ& or DNP-BSA plus 10 kmol/L of adenosine ( * different from comparable control values, are means k SEM of values from four experiments;
p < 0.05.
myo[*-3H]inositol, a 30-minute wash in buffer containing 10 mmol/L of inositol, and then washed twice in Tyrode’s buffer. The cells were challenged for 5 to 10 minutes with the approptiate agent, the reactions were stopped with 200 ~1 of ice-cold 15% trichloroacetic acid, and then centrifuged to collect thle supematant fraction. Ether extractions were performed, followed by neutralization with 5.6 mmol/L of borax, as detailed elsewhere.‘* Labeled inositol phosphates were separated with Dowex 1-8X resin (Bio-Rad Laboratories, Richmond, Calif.) columns to assess individual incorporation cofradiolahel.
Statistical
analysis
of data
Results a~ presented as means 2 SEM for three or more experiments performed in duplicate. Statistical significance was calculated with the paired, two-tailed Student’s t test unless it is otherwise noted.
Material The following materials were purchased from the manufacturers indicated: adenosine, aprotinin, leupeptin, phorbol myristate, oleic acid, acetate, N-acetyl-j3-D-glucosamide, 2-mercaptoethanol, phenylmethylsulfonyl fluoride, L-ol-lyso-PC, oleoyl, and myristoyl, palmitoyl, and lauroyl derivatives of lyso-PC from Sigma Chemical Co.; [y-‘*P]ATP from Amersham, Arlington Heights, Ill.; lysolecithin, L-cy-lyso-PC, oleoyl, and myristoyl forms from Avanti Polar Lipids, Pelham, Ala.; calcium ionophore, A23187, pertussis toxin, and staurosporine from Calbiochem, La Jolla, Calif.; [‘H]LTC, and RIA kit from New
England Nuclear, Boston, Mass.; tissue-culture media and supplements, PKC assay system, BAPl’A tetraacetoxymethy1 ester from GIBCO, Grand Island, N.Y.; and fura 2 from Molecular Probes, Eugene, Ore. The following materials were generously donated: mouse hybridoma anti-DNP IgE antibody and DNP-BSA Ag (Drs. Futong Liu and David Katz, Quidel, La Jolla, Calif.).
RESULTS The ability of lyso-PC to modulate the release of preformed, granule-associated mast cell mediators was studied by incubating mouse bone marcow-derived mast cells for 20 minutes with various concentrations of lyso-PC, followed by the addition of a secretagogue in the absence or presence of exogenous adenosine. Mast cells incubated for 20 minutes with I- 10 kmol / L of lyso-PC exhibited a dose-dependent, statistically significant increase in @-hexosaminidase release compared to untreated control cells (Figs. 1 and 2). When specific Ag was used as the secretagogue, >5 pmol/L of lyso-PC significantly augmented P-hexosaminidase release compared to Ag alone (p < 0.05), and 10 p,mol/L of lyso-PC not only augmented preformed mediator release in cells stimulated with Ag and Ag plus 10 p,mol of adenosine, but also induced a substantial mediator release on its own (Fig. 1). With A23 187 to examine another mechanism of mast cell activation, lyso-PC performed equally well as a secretagogue but was not as effective
724
Marquardt
J. ALLERGY
and Walker
CLIN. IMMUNOL. NOVEMBER 1991
El Spontaneous l?Zd + A231 87 m + A23 & Ado
Qi
[LYSO PC] (/.A)
FIG. 2. Effect of lyso-PC on A23187-stimulated mast cell p-hexosaminidase release. Cells were treated the same as in Fig. 1 except that A23187 (1 kg/ml) was used as the secretagogue. Depicted are means 2 SEM of values from seven experiments; * different from control values, p < 0.005; ** increased f3-hexosaminidase release over comparable spontaneous release values, p < 0.01. All cell populations stimulated with A23187 and 10 pmol/L of adenosine released significantly more p-hexosaminidase than cells stimulated with A23187 alone, p < 0.05.
in enhancing A23 187~stimulated P-hexosaminidase release with or without the exogenous addition of adenosine (Fig. 2). The time course of the ability of lyso-PC to induce mast cell mediator release demonstrated a relatively slow activation of granule-associated mediator secretion that plateaued between 10 and 20 minutes (Fig. 3). In all cases in which lysoPC was present in the incubation, exogenous adenosine demonstrated only a minimal ability to potentiate further granule-associated mediator release. Examination of cells exposed to 10 bmol/L of lyso-PC revealed a consistent ability to exclude trypan blue and a leak of LDH at 20 minutes comparable to control cells (data not presented), suggesting that these concentrations are not cytotoxic under the experimental conditions described. Because of the interesting finding that lyso-PC, a natural product of mast cell activation, itself acted as a secretagogue, the ability of adenosine to potentiate secretion induced by lyso-PC was examined. Adenosine (10 pm01 / L) modestly potentiated the release of mast cell B-hexosaminidase induced by high concentrations of lyso-PC (20 p.mol/L) when it was added simultaneously with lyso-PC, but the magnitude of its augmentation was clearly less than that observed when A23187 or DNP Ag were used to activate the cells, and concentrations of lyso-PC > 10 PmollL appeared to exhibit some cytotoxic properties (Fig. 4).
Because the secretion of mast cell granule contents does not always correlate with the generation of leukotrienes, the effects of lyso-PC on mast cell LTC, production were examined. Lyso-PC induced a dosedependent production of LTC, with little effect on A23 187-stimulated LTC, production (Fig. 5). The presence of 10 p,mol/L of adenosine did not alter mast cell LTC, generation, consistent with past demonstrations of the inability of adenosine to alter newly generated mediator release. l6 In an attempt to understand the mechanism whereby lyso-PC induces mediator release, direct measurements of PKC activity were performed by assessing the phosphorylation of specific PKC substrate. Mast cells incubated for 5 minutes with 10 p,mol/L of lysoPC exhibited markedly increased membrane PKC activity similar to activity observed after stimulation with PMA or specific Ag (Fig. 6). To determine whether the activation of PKC by lyso-PC is necessary to induce mast cell secretion, staurosporine, a putative PKC inhibitor, was added to cell suspensions at the same time as the lyso-PC (Fig. 7). Staurosporine (1 pmol/L) significantly reduced the ability of specific Ag to stimulate mast cell degranulation without altering the spontaneous release of P-hexosaminidase. Stauropsorine partially inhibited the ability of A23 187 to induce P-hexosaminidase release but did not alter the mediator release stimulated by 10 pm01 / L of lyso-
VOLUME NUMBER
Lysophosphatidylcholine
BE 5
and
mast
cells
725
TIME (minutes) FIG. 3. Time course of release of f3-hexosaminidase from lyso-PC-stimulated mast cells. were challenged with 10 pmol/L of lyso-PC for times illustrated followed by centrifugation assay for pellet and supernatant f3-hexosaminidase. Depicted are means f SEM of values four experiments.
Cells and from
0 Spontaneous E2zd + lOhuM adenosine
0
1
[LYSO p”c] (rM;O
’
FIG. 4. Release of @hexosaminidase from lyso-PC-stimulated mast cells in the presence and absence of adenosine. Cells were incubated for 10 minutes with concentrations of iyso-PC illustrated in the absence (0) or presence (WI of 10 pmol/L of adenosine. Depicted are means f SEM of values from four experiments; * different from control values, p < 0.025; ** significant increase in p-hexosaminidase released compared to values in the absence of adenosine.
PC, suggesting that, although lyso-PC activated PKC, this activity is not the basis for its ability to induce mast cell secretion. To explore alternate mechanisms of action of lysoPC on mast cell secretion, the modulation of calcium
fluxes by lyso-PC was studied with the probe, fura 2. Resting mast cells contained a relatively low concentration of ionized calcium that was significantly increased within seconds after stimulation with specific Ag (Fig. 8) and slowly returned toward baseline dur-
726
Marquardt
and Walker
J. ALLERGY
CLIN. IMMUNOL. NOVEMBER 1991
I Spontaneous EZZJ + A23187 EiSl + A23187 & Ado
0
5.0
1.0
[LYSO PC] (@vt) FIG. 5. Effect of lyso-PC for 20 minutes with 0 to or A23187 and 10 pmol/L by radioimmunoassay. creased LTC, compared
-
700
i*-
600
z E a i3 % s >r .e ‘Z> Y V
E
on A23187stimulated mast cell LTC, generation. Cells were incubated 10 pmol/L of lyso-PC and challenged with buffer (II), A23187 (@ZI), of adenosine ( ) for 20 minutes. Supernatant LTC, was quantitated Depicted are means + SEM of values from three experiments; * into controls, p < 0.05.
0 cytoso1 l?iZ Membrone 1
500 400 300 200 100 0 control
lyso
PC
PMA
antigen
FIG. 6. Assessment of cytosolic (0) and membrane (fZ!I) PKC activities in mast cells stimulated with buffer alone (control), 10 pmol/L of lyso-PC, 0.1 wmol/L of PMA, or 200 ng of DNP-BSA Ag for 5 minutes. =P counts per minute incorporated into specific PKC substrate in the presence of pseudosubstrate were subtracted from 32P cpm incorporated into specific PKC substrate in the absence of pseudosubstrate to determine PKC specific picomoles per minute of enzyme activity. Membrane PKC activity was significantly greater than control values with all stimuli (p < 0.05). Illustrated are means +. SD of values from three experiments.
ing the next 10 minutes. The additional presence of 10 pmol/L of adenosine enhanced the magnitude of this increase and maintained the elevated calcium concentrations for up to 10 minutes. Lyso-PC (10 pmol/L) induced a slow, steady enhancement of intracellular free-calcium levels that began to plateau in
6 to 10 minutes, and the addition of exogenous adenosine modestly augmented these calcium concentrations (Fig. 8). To determine the source of the observed increase in intracellular free calcium in lyso-PC stimulated cells, cells were incubated for 40 minutes in buffer of BAPTA tetraacetoxymethyl ester to chelate
VOLUME NUMBER
88 5
Lysophosphatidylcholine
0 BY
and
mast
cells
727
Control + Staurosporine
Control
Antigen
A231
87
Lyso
PC
FIG. 7. Effect of staurosporine on mast cell f3-hexosaminidase release induced by Ag, A23187, or 10 pmol/L of lyso-PC. Mast cells were incubated for 5 minutes with buffer alone 0 or 1 pmol/L of staurosporine WI and challenged with the above secretagogues for 10 minutes; * decreased %-hexosaminidase release compared to values in the absence of staurosporine, p < 0.01, calculated from six experiments.
-0 -0 -a ft -A
Lyso PC Lyso PC + Ado Antigen Antigen + Ado
.-A--.------:p’-Z I /O/O’
A@/
Y/O /\ 0
A-
A-A
-A
A
100
1
! 0
2
4
TIME
6
10
(minutes)
FIG. 8. Effect of lyso-PC on intracellular free-calcium levels. Cells were ionized calcium levels in Ag-stimulated and 10 pmol/L of lyso-PC-stimulated and presence of 10 pmol/L of adenosine were assessed continuously Depicted are means of values from two or more experiments.
intracellular calcium. This treatment abrogated the ability of AG to induce P-hexosaminidase release but had no effect on lyso-PC-induced mediator release (Fig. 9). However, chelation of extracellular calcium with 1 mmol/L of EGTA suppressed phexosaminidase release induced by either Ag or lyso-
8
loaded with fura 2, and cells in the absence for at least 10 minutes.
PC, suggesting that the increase in intracellular calcium by lyso-PC is due to an influx of calcium from the extracellular milleau. The roles of phospholipase C activation and G proteins in the mechanism of action of lyso-PC on mast cell secretion were partially addressed in two series
728
Marquardt
and Walker
J. ALLERGY
0 ezil !m
CLIN. IMMUNOL. NOVEMBER 1991
Control + Antigen + Lyso PC
medium
BAPTA-AM
EGTA
FIG. 9. Effect of intracellular (BAPTA-acetoxymethyl ester, 0.25 mmol/L) and extracellular (EGTA, 1 mmol/L) calcium chelators on mast cell 5-hexosaminidase release. Mast cells were preincubated for 30 minutes with BAPTA-acetoxymethylester or buffer alone and challenged with buffer (0). DNP-BSA Ag (KZ?I), or 10 pmol/L of lyso-PC ( 1. Some untreated cells were challenged with the same secretagogues in buffer containing EGTA. Illustrated are means -+ SD of values from three experiments.
of experiments. Mast cell activation by lyso-PC did not result in an increase in inositol trisphosphate production (data not presented), demonstrating a difference from Ag-induced mast cell secretion. Preincubation with pertussis toxin at concentrations previously demonstrated to adenosine diphosphateribosylate, the Gi protein, failed to inhibit the release of l3-hexosaminidase release induced by lyso-PC, although a modest inhibition of Ag-induced P-hexosaminidase release was observed” (Fig. 10). DISCUSSION
Lyso-PC is a natural product of the action of phospholipase A2 on phosphatidylcholine. Lyso-PC at concentrations 30 kmol/L of 1ys0-PC.~ Analogs of diacylglycerol, a natural product of the action of phospholipase C on phosphatidylinositols, stimulate phosphatidylcholine metabolism and a rapid increase in lyso-PC in rat pituitary cells by a mechanism distinct from the activation of PKC.” Furthermore, lysoPC and diacylglycerols themselves are membrane fusogens that may promote the secretory process in certain cell types.3 Therefore, in theory, lyso-PC has the potential to modulate mast cell intracellular biochemical processes in a number of ways. In exploring the role of exogenous lyso-PC in altering mast cell granule-associated mediator release,
it was observed that low concentrations of lyso-PC (
Hyper-IgE
88 5
18. Sampson HA, Buckley RH. Human IgE synthesis in vitro: reassessment.J Immunol 1981;127:829-34. 19. Gleich GJ, Averbeck AK, Svedlund HA. Measurement of IgE in normal and allergic serum by radioimmunoassay. J Lab Clin Med 1971;77:690-8. 20. Vercelli D, Jabara HH, Cunningham-Rundles C, et al. Regulation of immunoglobulin (1g)E synthesis in the hyper-IgE syndrome. J Clin Invest 1990;85:1666-71. 21. King CL, Gallin JI, Malech HL, Abramson SL, Nutman TB. Regulation of immunoglobulin production in hyperimmunoglobulin-E recurrent-infection syndrome by interferon-y. Proc Nat1 Acad Sci USA 1989;86: 10085-g. 22. Del l%:te G, Tiri A, Maggi E, et al. Defective in vitro production of y-interferon and tumor necrosis factor by circulating T cells from patients with the hyperimmunoglobulin E syndrome. J Clin Invest 1989;84:1830-5. 23. Finkelman FD, Holmes J, Urban JF Jr, Paul WE, Katona IM. T help requirements for the generation of an in vivo IgE response a late acting form of T cell help other than IL-4 is required for IgE but not for IgGl production. J Immunol 1989;1,12:403-8.
B cells
are refractory
to IL-4
24. Vercelli D, Jabara HH, Arai K-I, Geha RS. Induction of human IgE synthesis requires interleukin-4 and T/B cell interactions involving the T cell receptor/CD3 complex and MHC class II antigens. J Exp Med 169: 1295-1307. 25. Li JTC, Yunginger JW, Reed CE, Jaffe HS, Nelson DR, Gleich GJ. Lack of suppression of IgE production by recombinant interferon gamma: a controlled trial in patients with allergic rhinitis. J ALLERGYCLIN IMMUNOL 1990;85:934-40. 26. Boguniewicz M, Jaffe HS, Izu A, et al. Recombinant gamma interferon in treatment of patients with atopic dermatitis and elevated IgE levels. Am J Med 1990;88:365-70. 27. Gamkrelidze A, Bjorksten B. The determination of IgE synthesis in vitro: methodological aspects. J Immunol Methods 1990;130:9-13. 28. Massicot JG, Ishizaka K. Workshop on measurement of in vitro IgE synthesisand regulation of IgE synthesis.J ALLERGY CLIN IMMUNOL 1986;77:544-54. 29. King CL, Poindexter RW, Ragunathan J, et al. Frequency analysis of IgE-secreting B-lymphocytes in persons with normal or elevated serum IgE levels. J Immunol 1991;146:147883.
Lysophosphatidylcholine induces mast cell secretion and protein kinase C activation Diana L. Marquardt,
MD, and Linda L. Walker, BS San Diego, Calif.
Lysophosphatidylcholine (lyso-PC), a natural product of phospholipase A, activity, induced the secretion of both granule-associated @hexosaminidase and newly generated leukotriene C, from mouse bone marrow-derived mast cells. Micromolar concentrations of lyso-PC potentiated the release of /3-hexosaminidase induced by specific antigen but not the calcium ionophore, A23187. Exogenous adenosine was relatively ineffective in enhancing P-hexosaminidase release from cells challenged with lyso PC. Lyso-PC caused a marked increase in intracellular free-calcium levels and induced the activation of protein kinase C (PKC). These effects could not be abrogated by a prolonged preincubation with pertussis toxin. Staurosporine. an inhibitor of PKC, partially inhibited the abilities of antigen and A23187 to induce P-hexosaminidase release but was ineffective when lyso-PC was the secretagogue. Lyso-PC appears to activate mast cell PKC, but its ability to stimulate mast cell mediator release appears to be related to its abiliv to elevate intracellular free calcium concentrations. (J ALLERGY CLIN IMMUNOL 1991;88:721-30.) Key words: Mast cells, protein kinase C. staurosporine, lysophosphatidylcholine
From the Department of Medicine, University of California-San Diego School of Medicine, San Diego, Calif. Supported by National Institutes of Health Grants AI-17268 and AI-25507, and the Hartford Foundation. Received for publication May 31, 1990. Revised June 3, 1991. Accepted for publication June 6, 1991. Reprint requests: Diana Marquardt, MD, c/o UCSD Medical Center, 8417, 225 Dickinson St., San Diego, CA 92103. l/1/31505
pertussis toxin, calcium,
Investigation of the biochemical steps necessary to support the release of mediators from mouse bone marrow-derived mast cells has led to the conclusion that the interactions between various receptors, G proteins, phospholipases , and protein kinases are all parts of this complex signal transduction system. At least three phospholipases have been studied extensively in various secretory cells and tissues. Phospholipase D induces the release of phosphatidic acid from phos721
722
Marquardt
and Walker
Abbreviations
Lyso-PC: DNP: LDH: BSA: BAPTA: Ag: LT: EGTA: j2P: PMA: ATP: PKC:
used
Lysophosphatidylcholine Dinitrophenyl Lactic dehydrogenase Bovine serumalbumin Bis-(o-aminophenoxy-)-ethaneN,N,N’,N’,-tetraacetic acid Antigen Leukotriene Ethyleneglycol-bis-(B-aminoethylether)N,N,N’,N’,-tetraaceticacid Radioactiveisotopeof phosphorus Phorbolmyristateacetate Adenosinetriphosphate ProteinkinaseC
pholipids, and the action of phosphatidic acid phosphohydrolase on phosphatidic acid may contribute to the production of diacylglycerol, a membrane fusogen and transient natural activator of PKC. ’ Phospholipase C activation may induce the production of diacylglycerol and inositol trisphosphates.’ Phospholipase A2 activation contributes to the production of free arachidonic acid, which may be metabolized to leukotrienes or prostaglandins, and lyso-PC, a potent membrane fusogen.3 Lyso-PC also has been reported to activate PKC over a narrow concentration range in selected cells and tissues.4. 5 Adenosine has been a modulator of mast cell secretion of particular interest because it appears to potentiate the release of mast cell mediators through the stimulation of phospholipase C activity,6 resulting in the production of natural PKC activators.’ Adenosine augments the ongoing release of mast cell granule constituents, such as histamine or P-hexosaminidase, but fails to induce mediator release on its own and fails to augment the production of LTC,.’ Therefore, it is one of the relatively small number of agents that have been reported to affect preformed mediator release but not new mediator generation. Because agents that alter PKC activity have been demonstrated to alter the ability of mast cells to respond to the mediator release enhancing effects of adenosine9 and because lyso-PC is a natural product of phospholipase A2 activation during mast cell degranulation, an investigation of the ability of possible physiologic concentrations of lyso-PC to modulate mast cell adenosine responsiveness, activate PKC , and alter the mast cell secretory process was undertaken.
J. ALLERGY
MATERIAL Cell-culture
CLIN. IMMUNOL. NOVEMBER 1991
AND METHODS conditions
Bone marrowfrom femursof BALB/c mice (Simonsen Laboratories,Gilroy, Calif.) wasculturedin 20%WEHIsupematantas a sourceof interleukin-3” and RPM1containing 10%fetal calf serum,0.1 mmol/L of nonessential aminoacids,50 pmol/L of 2-mercaptoethanol, 2 mmol/L of L-glutamine,100pg/ml of streptomycin,and 100U/ml of penicillin. With weekly cell passage, by 21 daysof culture, cellswere>90% viable puremastcells.
Assays The releaseof B-hexosaminidase from stimulatedmast cells was assessed as previously describedin detail.” Briefly, cells werepassivelysensitizedwith anti-DNPIgE (1 Kg/ lo6of cells)andchallenged for 10minuteswith DNPBSA Ag (200 ng13 x 10’ cells), or 1 pg/ml of A23187, and the B-hexosaminidase presentin the resultingsupernatantsandcell pelletswasquantitatedby the hydrolysisof P-nitrophenyl-B-o-glucosaminide substrate.L’ Mast cell supematant LTC, concentrations wereassessed in experiments similarto thoseexperiments above, followed by the useof a [‘H]LTC, RIA kit. Somecross-reactivity betweenLTC,, LTD,, and LTE, hasbeendescribedin this RIA, but mousebone marrow-derived mast cells primarily synthesizeLTC, by lipoxygenationof arachidonic acid.I2 PKC activity wasmeasured by the phosphorylation of a syntheticpeptidefrom myelin basicproteinthat can act as a specificsubstratefor PKC.” Mast cells(2 X 106/250~1) were challengedwith appropriateagentsfor 5 minutesat 37” C, and reactionswere terminatedby the additionof two equal volumesof cold buffer (40 mmol/L of Tris, 1 mmol/L of ethylenediaminetetraacetic acid, 1 mmol/L of EGTA, and 50 kg/ml eachof aprotininand leupeptin, pH 7.5), sonicationfor 10 seconds,and centrifugationat 100,000g for 30minutesat 4” C. Supematants weresaved, and pellets were resuspended in 0.5 ml of buffer (20 mm01 /L of Tris, 0.5 mm01 IL of ethylenediaminetetraacetic acid, 0.5 mmol/L of EGTA, 0.5% Triton X-100, and 25 pg/ml eachof aprotininandleupeptin,pH 7.5) andsonicated again for 10 seconds.Aliquots of supematantand pelletfractions(5 pl) wereassayed by preincubationfor 20 minutesat roomtemperaturein the absence or presenceof a pseudosubstrate inhibitor, the additionof 32PgammaATP and substrateat 30” C for 5 minutes,andspottingsamples on phosphocellulose disksand determiningthe 3zPcounts in the absenceandpresenceof the inhibitor. Intracellularfree-calciumlevelswerequantitatedby loading cellswith the fura 2 probeandmeasuringfura 2 fluorescence,aspreviouslydescribedfor this mastcell population.‘, I4 LDH activitiesin cell supematants, pellets,andsonicated pelletswereassessed with a calorimetrickit purchased from SigmaChemicalCo., St. Louis, MO. The productionof the inositolphosphates, IP, IPI, and IP,, wasdeterminedby incubatingcellsfor 18 hourswith
VOLUME NUMBER
Lysophosphatidylcholine
88 5
0 Spontaneous IZI + Antigen gzfds + Ag &Ado
and
mast
ceils
723
* *
* 1
FIG. 1. Effect of lyso-PC on Ag-stimulated mast cell P-hexosaminidase release. Cells were incubated for 20 minutes with 0 to 10 pmol/L of lyso-PC and challenged with buffer (U), DNP) for 10 minutes. Depicted BSA Ag (200 ng) (tZZ& or DNP-BSA plus 10 kmol/L of adenosine ( * different from comparable control values, are means k SEM of values from four experiments;
p < 0.05.
myo[*-3H]inositol, a 30-minute wash in buffer containing 10 mmol/L of inositol, and then washed twice in Tyrode’s buffer. The cells were challenged for 5 to 10 minutes with the approptiate agent, the reactions were stopped with 200 ~1 of ice-cold 15% trichloroacetic acid, and then centrifuged to collect thle supematant fraction. Ether extractions were performed, followed by neutralization with 5.6 mmol/L of borax, as detailed elsewhere.‘* Labeled inositol phosphates were separated with Dowex 1-8X resin (Bio-Rad Laboratories, Richmond, Calif.) columns to assess individual incorporation cofradiolahel.
Statistical
analysis
of data
Results a~ presented as means 2 SEM for three or more experiments performed in duplicate. Statistical significance was calculated with the paired, two-tailed Student’s t test unless it is otherwise noted.
Material The following materials were purchased from the manufacturers indicated: adenosine, aprotinin, leupeptin, phorbol myristate, oleic acid, acetate, N-acetyl-j3-D-glucosamide, 2-mercaptoethanol, phenylmethylsulfonyl fluoride, L-ol-lyso-PC, oleoyl, and myristoyl, palmitoyl, and lauroyl derivatives of lyso-PC from Sigma Chemical Co.; [y-‘*P]ATP from Amersham, Arlington Heights, Ill.; lysolecithin, L-cy-lyso-PC, oleoyl, and myristoyl forms from Avanti Polar Lipids, Pelham, Ala.; calcium ionophore, A23187, pertussis toxin, and staurosporine from Calbiochem, La Jolla, Calif.; [‘H]LTC, and RIA kit from New
England Nuclear, Boston, Mass.; tissue-culture media and supplements, PKC assay system, BAPl’A tetraacetoxymethy1 ester from GIBCO, Grand Island, N.Y.; and fura 2 from Molecular Probes, Eugene, Ore. The following materials were generously donated: mouse hybridoma anti-DNP IgE antibody and DNP-BSA Ag (Drs. Futong Liu and David Katz, Quidel, La Jolla, Calif.).
RESULTS The ability of lyso-PC to modulate the release of preformed, granule-associated mast cell mediators was studied by incubating mouse bone marcow-derived mast cells for 20 minutes with various concentrations of lyso-PC, followed by the addition of a secretagogue in the absence or presence of exogenous adenosine. Mast cells incubated for 20 minutes with I- 10 kmol / L of lyso-PC exhibited a dose-dependent, statistically significant increase in @-hexosaminidase release compared to untreated control cells (Figs. 1 and 2). When specific Ag was used as the secretagogue, >5 pmol/L of lyso-PC significantly augmented P-hexosaminidase release compared to Ag alone (p < 0.05), and 10 p,mol/L of lyso-PC not only augmented preformed mediator release in cells stimulated with Ag and Ag plus 10 p,mol of adenosine, but also induced a substantial mediator release on its own (Fig. 1). With A23 187 to examine another mechanism of mast cell activation, lyso-PC performed equally well as a secretagogue but was not as effective
724
Marquardt
J. ALLERGY
and Walker
CLIN. IMMUNOL. NOVEMBER 1991
El Spontaneous l?Zd + A231 87 m + A23 & Ado
Qi
[LYSO PC] (/.A)
FIG. 2. Effect of lyso-PC on A23187-stimulated mast cell p-hexosaminidase release. Cells were treated the same as in Fig. 1 except that A23187 (1 kg/ml) was used as the secretagogue. Depicted are means 2 SEM of values from seven experiments; * different from control values, p < 0.005; ** increased f3-hexosaminidase release over comparable spontaneous release values, p < 0.01. All cell populations stimulated with A23187 and 10 pmol/L of adenosine released significantly more p-hexosaminidase than cells stimulated with A23187 alone, p < 0.05.
in enhancing A23 187~stimulated P-hexosaminidase release with or without the exogenous addition of adenosine (Fig. 2). The time course of the ability of lyso-PC to induce mast cell mediator release demonstrated a relatively slow activation of granule-associated mediator secretion that plateaued between 10 and 20 minutes (Fig. 3). In all cases in which lysoPC was present in the incubation, exogenous adenosine demonstrated only a minimal ability to potentiate further granule-associated mediator release. Examination of cells exposed to 10 bmol/L of lyso-PC revealed a consistent ability to exclude trypan blue and a leak of LDH at 20 minutes comparable to control cells (data not presented), suggesting that these concentrations are not cytotoxic under the experimental conditions described. Because of the interesting finding that lyso-PC, a natural product of mast cell activation, itself acted as a secretagogue, the ability of adenosine to potentiate secretion induced by lyso-PC was examined. Adenosine (10 pm01 / L) modestly potentiated the release of mast cell B-hexosaminidase induced by high concentrations of lyso-PC (20 p.mol/L) when it was added simultaneously with lyso-PC, but the magnitude of its augmentation was clearly less than that observed when A23187 or DNP Ag were used to activate the cells, and concentrations of lyso-PC > 10 PmollL appeared to exhibit some cytotoxic properties (Fig. 4).
Because the secretion of mast cell granule contents does not always correlate with the generation of leukotrienes, the effects of lyso-PC on mast cell LTC, production were examined. Lyso-PC induced a dosedependent production of LTC, with little effect on A23 187-stimulated LTC, production (Fig. 5). The presence of 10 p,mol/L of adenosine did not alter mast cell LTC, generation, consistent with past demonstrations of the inability of adenosine to alter newly generated mediator release. l6 In an attempt to understand the mechanism whereby lyso-PC induces mediator release, direct measurements of PKC activity were performed by assessing the phosphorylation of specific PKC substrate. Mast cells incubated for 5 minutes with 10 p,mol/L of lysoPC exhibited markedly increased membrane PKC activity similar to activity observed after stimulation with PMA or specific Ag (Fig. 6). To determine whether the activation of PKC by lyso-PC is necessary to induce mast cell secretion, staurosporine, a putative PKC inhibitor, was added to cell suspensions at the same time as the lyso-PC (Fig. 7). Staurosporine (1 pmol/L) significantly reduced the ability of specific Ag to stimulate mast cell degranulation without altering the spontaneous release of P-hexosaminidase. Stauropsorine partially inhibited the ability of A23 187 to induce P-hexosaminidase release but did not alter the mediator release stimulated by 10 pm01 / L of lyso-
VOLUME NUMBER
Lysophosphatidylcholine
BE 5
and
mast
cells
725
TIME (minutes) FIG. 3. Time course of release of f3-hexosaminidase from lyso-PC-stimulated mast cells. were challenged with 10 pmol/L of lyso-PC for times illustrated followed by centrifugation assay for pellet and supernatant f3-hexosaminidase. Depicted are means f SEM of values four experiments.
Cells and from
0 Spontaneous E2zd + lOhuM adenosine
0
1
[LYSO p”c] (rM;O
’
FIG. 4. Release of @hexosaminidase from lyso-PC-stimulated mast cells in the presence and absence of adenosine. Cells were incubated for 10 minutes with concentrations of iyso-PC illustrated in the absence (0) or presence (WI of 10 pmol/L of adenosine. Depicted are means f SEM of values from four experiments; * different from control values, p < 0.025; ** significant increase in p-hexosaminidase released compared to values in the absence of adenosine.
PC, suggesting that, although lyso-PC activated PKC, this activity is not the basis for its ability to induce mast cell secretion. To explore alternate mechanisms of action of lysoPC on mast cell secretion, the modulation of calcium
fluxes by lyso-PC was studied with the probe, fura 2. Resting mast cells contained a relatively low concentration of ionized calcium that was significantly increased within seconds after stimulation with specific Ag (Fig. 8) and slowly returned toward baseline dur-
726
Marquardt
and Walker
J. ALLERGY
CLIN. IMMUNOL. NOVEMBER 1991
I Spontaneous EZZJ + A23187 EiSl + A23187 & Ado
0
5.0
1.0
[LYSO PC] (@vt) FIG. 5. Effect of lyso-PC for 20 minutes with 0 to or A23187 and 10 pmol/L by radioimmunoassay. creased LTC, compared
-
700
i*-
600
z E a i3 % s >r .e ‘Z> Y V
E
on A23187stimulated mast cell LTC, generation. Cells were incubated 10 pmol/L of lyso-PC and challenged with buffer (II), A23187 (@ZI), of adenosine ( ) for 20 minutes. Supernatant LTC, was quantitated Depicted are means + SEM of values from three experiments; * into controls, p < 0.05.
0 cytoso1 l?iZ Membrone 1
500 400 300 200 100 0 control
lyso
PC
PMA
antigen
FIG. 6. Assessment of cytosolic (0) and membrane (fZ!I) PKC activities in mast cells stimulated with buffer alone (control), 10 pmol/L of lyso-PC, 0.1 wmol/L of PMA, or 200 ng of DNP-BSA Ag for 5 minutes. =P counts per minute incorporated into specific PKC substrate in the presence of pseudosubstrate were subtracted from 32P cpm incorporated into specific PKC substrate in the absence of pseudosubstrate to determine PKC specific picomoles per minute of enzyme activity. Membrane PKC activity was significantly greater than control values with all stimuli (p < 0.05). Illustrated are means +. SD of values from three experiments.
ing the next 10 minutes. The additional presence of 10 pmol/L of adenosine enhanced the magnitude of this increase and maintained the elevated calcium concentrations for up to 10 minutes. Lyso-PC (10 pmol/L) induced a slow, steady enhancement of intracellular free-calcium levels that began to plateau in
6 to 10 minutes, and the addition of exogenous adenosine modestly augmented these calcium concentrations (Fig. 8). To determine the source of the observed increase in intracellular free calcium in lyso-PC stimulated cells, cells were incubated for 40 minutes in buffer of BAPTA tetraacetoxymethyl ester to chelate
VOLUME NUMBER
88 5
Lysophosphatidylcholine
0 BY
and
mast
cells
727
Control + Staurosporine
Control
Antigen
A231
87
Lyso
PC
FIG. 7. Effect of staurosporine on mast cell f3-hexosaminidase release induced by Ag, A23187, or 10 pmol/L of lyso-PC. Mast cells were incubated for 5 minutes with buffer alone 0 or 1 pmol/L of staurosporine WI and challenged with the above secretagogues for 10 minutes; * decreased %-hexosaminidase release compared to values in the absence of staurosporine, p < 0.01, calculated from six experiments.
-0 -0 -a ft -A
Lyso PC Lyso PC + Ado Antigen Antigen + Ado
.-A--.------:p’-Z I /O/O’
A@/
Y/O /\ 0
A-
A-A
-A
A
100
1
! 0
2
4
TIME
6
10
(minutes)
FIG. 8. Effect of lyso-PC on intracellular free-calcium levels. Cells were ionized calcium levels in Ag-stimulated and 10 pmol/L of lyso-PC-stimulated and presence of 10 pmol/L of adenosine were assessed continuously Depicted are means of values from two or more experiments.
intracellular calcium. This treatment abrogated the ability of AG to induce P-hexosaminidase release but had no effect on lyso-PC-induced mediator release (Fig. 9). However, chelation of extracellular calcium with 1 mmol/L of EGTA suppressed phexosaminidase release induced by either Ag or lyso-
8
loaded with fura 2, and cells in the absence for at least 10 minutes.
PC, suggesting that the increase in intracellular calcium by lyso-PC is due to an influx of calcium from the extracellular milleau. The roles of phospholipase C activation and G proteins in the mechanism of action of lyso-PC on mast cell secretion were partially addressed in two series
728
Marquardt
and Walker
J. ALLERGY
0 ezil !m
CLIN. IMMUNOL. NOVEMBER 1991
Control + Antigen + Lyso PC
medium
BAPTA-AM
EGTA
FIG. 9. Effect of intracellular (BAPTA-acetoxymethyl ester, 0.25 mmol/L) and extracellular (EGTA, 1 mmol/L) calcium chelators on mast cell 5-hexosaminidase release. Mast cells were preincubated for 30 minutes with BAPTA-acetoxymethylester or buffer alone and challenged with buffer (0). DNP-BSA Ag (KZ?I), or 10 pmol/L of lyso-PC ( 1. Some untreated cells were challenged with the same secretagogues in buffer containing EGTA. Illustrated are means -+ SD of values from three experiments.
of experiments. Mast cell activation by lyso-PC did not result in an increase in inositol trisphosphate production (data not presented), demonstrating a difference from Ag-induced mast cell secretion. Preincubation with pertussis toxin at concentrations previously demonstrated to adenosine diphosphateribosylate, the Gi protein, failed to inhibit the release of l3-hexosaminidase release induced by lyso-PC, although a modest inhibition of Ag-induced P-hexosaminidase release was observed” (Fig. 10). DISCUSSION
Lyso-PC is a natural product of the action of phospholipase A2 on phosphatidylcholine. Lyso-PC at concentrations 30 kmol/L of 1ys0-PC.~ Analogs of diacylglycerol, a natural product of the action of phospholipase C on phosphatidylinositols, stimulate phosphatidylcholine metabolism and a rapid increase in lyso-PC in rat pituitary cells by a mechanism distinct from the activation of PKC.” Furthermore, lysoPC and diacylglycerols themselves are membrane fusogens that may promote the secretory process in certain cell types.3 Therefore, in theory, lyso-PC has the potential to modulate mast cell intracellular biochemical processes in a number of ways. In exploring the role of exogenous lyso-PC in altering mast cell granule-associated mediator release,
it was observed that low concentrations of lyso-PC (
