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Dexamethasone action inhibits the release of arachidonic acid from phosphatidylcholine during the suppression of yeast phagocytosis in macrophage cultures
Vol. 153, No. 2, 1988 June 16,1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 583-590
DEXAMETHASONE ACTION INHIBITS THE RELEASE OF ARACHIDONIC ACID FROM PHOSPHATIDYLCHOLINE DURING THE SUPPRESSION OF YEAST PHAGOCYTOSIS IN MACROPHAGE CULTURES
Jeanne L. Becker I*, Robert J. Grasso I and John S. Davis 2 Department of Medical Microbiology and Immunology I, Department of Internal Medicine 2, College of Medicine, University of South Florida Tampa, Florida, 33612 Received April 13, 1988
SUMMARY: Dexamethasone suppresses phagocytosis of heat killed Saccharomyces cerevisiae in cultures of murine peritoneal macrophages. Recent observations suggest that dexamethasone induces a phagocytic inhibitory protein that suppresses yeast ingestion by inhibiting macrophage phospholipase A2 activity. The present investigation, therefore, examined whether macrophage lipid metabolism is modulated by dexamethasone. Control and steroid treated macrophages were allowed to incorporate radiolabeled arachidonate and were incubated subsequently in the absence and presence of yeast. Following ingestion by control macrophages, arachidonate from phosphatidylcholine was readily cleaved to free fatty acid and transferred to the neutral lipid fraction. In contrast, arachidonate release was inhibited in dexamethasone treated macrophages. These results suggest that the suppression of yeast phagocytosis by dexamethasone action may be associated with the inhibition of phospholipase A2 activity. © 1988 Academic Press, Inc.
Glucocorticoid steroids are potent antiinflammatory and immunosuppressire agents that decrease host resistance to infectious diseases (1-3).
In
this regard, we have demonstrated that DEX and other glucocorticoids inhibit the ingestion of heat-killed Saccharomyces cerevisiae particles in cultures of murine resident and elicited peritoneal macrophages (4-9). The mechanism underlying this steroid induced suppressive effect on yeast phagocytosis remains to be elucidated.
In contrast to reversing
other glucocorticoid directed inhibitory responses by supplying exogenous AA (I0), phagocytic inhibitory responses in DEX-treated macrophage cultures are not reversible by supplying exogenous AA either alone or in combination with indomethacin and nordihydroguaiaretic acid (8).
More recent
*Present Address: H. Lee Moffitt Cancer Center and Research Institute, P.O. Box 280179, Tampa, FL 33682 DEX = Dexamethasone; PIP = Phagocytosis Inhibitory Protein; PLA2 = Phospholipase A2; TLC = Thin Layer Chromatography; A A = Arachidonic Acid; PA = Phosphatidic Acid; PE = Phosphatidylethanolamine; PI = Phosphatidylinositol; PC = Phosphatidylcholine; MAG = Monoacylglycerol (monopalmitoylglycerol); OH = Cholesterol; DAG = Diacylglycerol (diolein); TAG = Triacylglycerol (triolein); and BPB = p-Bromophenacyl Bromide. ABBREVIATIONS:
583
0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 153, No. 2, 1988
evidence indicates that
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DEX induces (via de nero RNA and protein synthesis)
a heat-labile, trypsin-sensitive factor termed PIP, which mediates the inhibition of yeast ingestion (9).
Since PIP activity is neutralized by
monoclonal antibodies directed against lipocortin (9), the antiphagocytic factor appears to belong to this family of PLA2 inhibitory proteins that are induced by glucocorticoids
(11-16).
Therefore, we postulate that glucocorticoid action inhibits PLA2 activity which, in some fashion, interferes with the normal functioning of macrophage membranes required for yeast phagocytosis to occur.
Although AA
itself, as well as its eicosanoid products, may not directly influence the DEX induced inhibition of phagocytosis, this suppressive response may be related to altered lipid metabolism. Our results suggest that PLA2 activity is suppressed
during the inhibition of phagocytosis in DEX treated macro-
phage cultures.
MATERIALS AND METHODS
Macrophage Cultures Cultures of resident peritoneal macrophages were established from leukocytes lavaged from male C57BL/6 mice as described in detail elsewhere (17). When the cultures were established, control macrophages were supplied culture medium (Dulbecco's Modified Eagle Medium supplemented with antibiotics and 10% heat-inactivated fetal bovine serum) lacking the steroid and the experimental macrophages received medium supplemented with i ~M DEX. For the yeast ph~gocytosis assays, each well of a 96 well microtiter plate contained 1.5xl0 adherent macrophages in 0.15 ml of medium. ~or the lipid studies, each well of a 24 well cluster plate contained 1.3x10 v adherent macrophages in 1.5 ml of mediumbC All control and steroid treated cultures were incubated for 3 days at 37 in an atmosphere of 95% air-5% CO 2 prior to initiating the experiments. Yeast Phagocytosis Assays Ingestion of [3H] S. cerevisiae by macrophages in the wells of the microtiter plates was ~ a s u r e d w~th a radiometric yeast phagocytosis assay (17). Phagocytic capacities of macrophages in replicate control and i ~M DEX treated cultures are defined as the mean CPM values + S.E.M. Radiolabeling and Extraction of Macrophage Lipids Three day old control and I}IM DEX t r ~ t e d macrophages were exposed for 4 hr at 37 °C to either i ~Ci/ml of [I-~C]-AA (spec. act. = 58 mCi/mmol) or 6.7 ~Ci/ml of [5,6,8,9,11,12,14, 15-3H] -AA (spec. act. = 220 mCi/mmol; Amershsm, Inc., Arlington Heights, IL). After removing unincorporated radioactivity, the cultures were supplied respective fresh media and incubated at 37 °C for an additional 15 min in the absence and presence of non-radioactive heat-killed S. cerevisiae. All media was removed after placing the cultures at 0°C,-Macropnages were scraped from each well into 3 ml of absolute methanol at 0°C, to which 6 ml of chloroform and 0.25 ml of 1 N HCI was added. The ratio of chloroform/methanol/HCl was 2/1/0.25. The solution was vortexed for 30 sec and centrifuged for i0 min at i000 x g at 4°C. The aqueous phase was discarded. This washing procedure was repeated sequentially with chloroform/methanol/0.1 N HCI followed by chloroform/methanol/0.01 N HCI (3/48/47 v/v). The organic phase containing the extracted lipids was stored overnight at -70°C. On the following day, the extracts
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Vol. 153, No. 2, 1988
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were placed at 0°C, centrifuged as described above, and any remaining aqueous phase was discarded. The extracts were dried under a stream of nitrogen and dissolved in chloroform/methanol/water at 75/25/2 (v/v). TLC of Macrophage Phospholipids Two dimensional Redi-Coat silica gel TLC plates containing 10% magnesium acetate (Supelco, Bellefonte, PA) were developed to within i cm of the top in methanol/water (2/3) containing 1% potassium oxalate. Thereafter, the plates were air dried and activated at ll0°C for 30 min. The TLC plates were developed in chloroform/acetone/methanol/acetic acid/water (40/15/13/12/8 v/v) to within 2 cm from their upper edges. The plates were air dried and the lipids were identified by exposure to iodine vapors. Sample lipids were compared to standards obtained from Sigma Chemical Co. (St. Louis, MO). The material was scraped from the plate and radioactivity in each band was measured by liquid scintillation spectrometry, TLC of Macrophage Neutral Lipids The TLC plates were activated at II0UC for 45 min and spotted with the macrophage lipid extracts. The TLC plates were first developed in ether/benzene/ethanol/acetic acid (40/50/2/2 v/v) to 5 cm from the top. The plates were then air dried and developed further in ether/hexane (6/94 v/v) to within 2 cm from the upper edges. After the plates were air dried, the neutral lipid containing bands were visualized, scored, collected by scraping, and radioactivity measured. Authentic neutral lipid standards were obtained from Sigma Chemical Co. RESULTS
Suppression of Yeast Ingestion by BPB Our hypothesis predicts that inhibiting PLA2 activity should inhibit yeast phagocytosis.
Hence, we measured the phagocytic capacities of
macrophages exposed to BPB, an inhibitor of PLA2 (18).
Dose response
studies revealed that i0 pM BPB suppressed yeast ingestion but was not toxic to the macrophages as determined by trypan blue dye exclusion tests. Accordingly, 3 day old control and i pM DEX treated macrophage cultures were incubated at 37°C for up to 45 min in the absence and presence of i0 ~MBPB.
Yeast phagocytosis assays, each lasting 15 min, were initiated at
the time the phospholipase inhibitor was supplied, and at 15 min intervals thereafter.
Figure I illustrates that BPB suppressed yeast ingestion
almost completely in control cultures after 45 min.
The steroid alone
elicited a 55% inhibitory response after 3 days and 15 min of treatment with BPB almost completed suppressed yeast in the DEX treated cultures. The initial rates of the inhibitory responses elicited by BPB were nearly parallel in the control and steroid treated cultures.
Separate experiments
with the phospholipase inhibitors, quinacrine (i0 ~/~) and tetracaine (i00 pM), yielded similar results (data not shown). Modulation of Macrophage Lipid Metabolism Since phospholipid turnover can be measured by changes in AA (19), we examined the distribution of radiolabeled AA among phospholipids and
585
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
- c
11
4\
o CONTROL
_Lx
10 9 8
~T " I ~
\
? ,.p.o 7 m
0.
01#M DEX A IO~M BPB • 1 0 ~ BPB+ 1.M DEX [] BACKGROUND
k
6
u
3 2 1
0
15
30
45
TIME OF EXPOSURE TO BPB Iminl FIGURE 1.
Time Course o f BPB I n h i b i t i o n
o f Yeast P h a g o c y t o s i s i n C o n t r o l
Dan - - - ~ T r e a t e d Macrophage Cultures. Murine resident peritoneal macrophages were incubated for 3 days in the absence and presence of 1 NM DEX. On day 3, the cultures were exposed to i0 ~M BPB for up to 45 min. Phagocytic capacities were measured over 15 min assay periods in both sets of cultures at the times indicated by the symbols, which represent mean CPM values + S.E.M. (N=3).
neutral lipids in 3 day old control and I NM DEX treated macrophages after 15 min incubation periods in the absence and presence of unlabeled yeast particles. Table 1 shows the distribution of radioactivity incorporated into phospholipids and neutral lipids.
Total CPM incorporated into lipids of
individual samples was variable.
In these studies, 8,074 + 3,122 CPM were
present in phospholipids in nonphagocytizing control cultures with 2,464 + 1,415 CPM in neutral lipids.
In noningesting DEX treated cultures, 4,457 +
965 CPM were present in phospholipids and 1,259 + 279 CPM in neutral lipids, thus suggesting that basal phospholipid [urnover was reduced in DEX treated macrophages.
Following yeast phagocytosis, control cultures
exhibited 15,051 + 4,525 CPM in phospholipids and 19,604 + 11,402 CPM in neutral lipids; DEX treated cultures had 9,728 + 4,984 CPM in phospholipids and 4,933 + 2,985 CPM in neutral lipids f o l l o w J g yeast ingestion. Phosp~olipids contained 82% of the total radioactivity in control macrophages not exposed to yeast with over 50% of the isotope found in PC. The extracted neutral lipid fraction contained 18% of the radioactivity in
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Vol. 153, No. 2, 1988
TABLE
i.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Dexamethasone Action Inhibits the Conversion of [14C] AA from PC to Neutral Lipids During Yeast Phagocytosis [14C] AA-labeled Lipids a
Macrophage Cultures
15 Min Exposure to Unlabeled Yeast
Phospholipids (% of Total) Neutral PC
PE+PA
PI
Otherb
Lipids c
Control
-
55 + 2
16 + 2
7 + i
4 + I
18 + 5
Control
+
27 + 4
16 + 2
3 + 1
5 + 1
49 + 9
i ~M D E X
-
47 + 3
19 + 3
5 + 2
7 + 1
22 + 1
i pM DEX
+
40 + 3
17 + 5
4 + I
9 + I
29 + 6
a b c
Mean Percentage + S.E.M. (N=3), based on total radioactivity of sample. This fraction contained PI mono-and diphosphates. See Table 2 for individual neutral lipids that were measured in separate experiments.
these noningesting macrophages.
When phagocytosis was stimulated by the
addition of yeast, the percentage of radioactivity in the PC fraction decreased by 28% and in the neutral lipid fraction increased by 31%.
In
contrast, when DEX treated cultures were exposed to yeast, only a 7% decrease in radioactivity in PC occurred, with a corresponding increase of 7% in the neutral lipids. On a percentage basis, no major changes in radioactivity were observed in the remaining phospholipid fractions in either control or steroid treated cultures, regardless of whether yeast was present or not. Table 2 reveals the results of separate experiments that show the distribution of radioactivity in the major neutral lipids in macrophage cultures.
In the absence of phagocytosis, the total radioactivity in
neutral lipids in control cultures was 36,926 + 10,652 CPM; DEX treated cultures contained 4,750 + 1,348 CPM in neutral lipids under this condition.
Following yeast ingestion, 36,994 + 17,585 CPM were present in
neutral lipids in control cultures and 21,486 + 3,643 CPM were present in neutral lipid extracted from steroid treated macrophages. to control macrophages in the absence of yeast,
Thus, relative
the steroid treated
macrophages incorporated only 13% as much [3H]-AA into neutral lipids. During yeast phagocytosis in control cultures, 19% increase was observed in
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TABLE 2. Distribution of [3H] AA in Neutral Lipids of Control and Glucocorticoid Treated Macrophages in the Absence and Presence of Yeast Phagocytosis [3H] AA-labeled Neutral Lipids (% of Total) a Macrophage
15 Min
Cultures
Exposure to Unlabeled Yeast
AA
TAG
DAG
CE
MAG
CH
Control
-
9 +
2
74 +
3
6 + I
8 + I
3 + i
1 + 0
Control
+
28 +
2
54 +
3
8 + 2
4 + 0
5 + 1
2 + i
i ~M DEX
-
23 .+ 12
. 52 + II .
9. + 0
6 + I
I + 0
1 ~M DEX
+
15 +
3 + I
I + 0
a
2
69 +
4
. 9 + i.
8 + I
5 + I
Mean Percentage + S.E.M. (N=3), based on total radioactivity of sample.
the portion of radioactivity associated with free AA. This effect was accompanied by a 20% decrease in the percentage of radioactivity associated with TAG.
In contrast, when DEX treated macrophages were exposed to yeast,
a minimal 8% decrease occurred in the percentage of radiolabeled free AA. This effect was accompanied by a 17% increase in the percentage of radioactive TAG.
No major changes occurred in the percentage of
radioactivity associated with DAG, CE, MAG and CH in either control or steroid treated macrophages,
regardless of whether they were exposed to
yeast.
DISCUSSION These results demonstrate that glucocorticoid action inhibits phagocytosis potentially by modulating lipid metabolism. cultures,
Relative to control
the turnover of PC is suppressed in DEX treated macrophages
exposed to yeast.
Although DEX treated macrophages
number of yeast particles
ingest only a limited
(4-6), higher CPM values were recovered when the
cells were allowed to ingest yeast; similar results also occurred in control cultures.
This finding may indicate that the radiolabeled AA, which
might otherwise he released into the culture fluids,
is readily reesterified
into cellular lipid during the process of phagocytosis.
However, the total
CPM present in DEX treated cultures was about 50% of control values and the net increase in phospholipid turnover induced by phagocytosis was much greater in control cultures.
Moreover,
the relative amounts of the radio-
labeled arachidonate associated with each of the lipid fractions changes very little in macrophages exposed to DEX, regardless of whether or not yeast is supplied to the treated cultures.
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Based upon our previous studies, we postulate that the alterations of lipid metabolism produced by DEX may be mediated by steroid induced PIP. The data collected on the radiolabeled lipids presented in Table i implicate PLA2 as a likely target for DEX action.
This interpretation is supported
by the results of the BPB kinetic experiments, as well as by experiments with the phospholipase inhibitors, quinacrine and tetracaine. others have
Furthermore,
demonstrated that tetracaine suppresses phagocytosis of sheep
erythrocytes by murine elicited macrophages (20). In cultures of non-steroid treated, non-phagocytizing macrophages,
the
distribution of AA among the various lipid fractions agrees very well with previous studies performed by others with murine peritoneal macrophages (19,21) and with rabbit alveolar macrophages (22).
A similar distribution
of AA in phospholipids is present in DEX treated macrophages that are not exposed to yeast, even though the total amounts of radioactivity associated with phospholipids are considerably reduced relative to the controls. The release of AA from PC during yeast phagocytosis by control macrophages reflects normally functioning membrane activity required for ingestion.
Studies with HL60 lymphoma cells have shown that a shift in AA from
PC to neutral lipids also occurs when these cells are stimulated with either the chemotactic peptide N-formyl Nle-Leu-Phe-Nle-Tyr-Lys or the calcium ionophore A23187 (23), both of which activate PLA2 (24,25).
In
addition, stimulation of murine peritoneal macrophages with zymosan results in the release of AA from PC with a corresponding increase of the fatty acid in neutral lipids (26).
Thus, exposing control macrophage cell mem-
branes to an external physical stimulus, such as yeast particles, may result in increased PLA2 activity accompanied by the release of the isotope from PC to AA. We also observed that control macrophages exhibit a decreased percentage of AA in TAG and corresponding increase in free AA following yeast ingestion.
These changes in arachidonate distribution in the neutral
lipids may be associated with the activation of TAG lipase, whose activity may be enhanced during phagocytosis (27).
In contrast, DEX treated macro-
phages exhibit an increased percentage of AA in TAG, which may reflect decreased levels of the activity of this enzyme. Taken together, the evidence presented in this report supports the hypothesis that glucocorticoid action inhibits phagocytosis by suppressing PLA2 activity, presumably via PIP activity.
This notion is strengthened by
the recent finding that glucocorticoids preferentially inhibit PLA2 within C62B glioma cells in vivo, but exert very little inhibitory effect on phospholipase C (28).
Whether this in fact also occurs in DEX treated
macrophages remains to be investigated.
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ACKNOWLEDGEMENTS
This research was supported in part by USPHS NIH Grant Number 1 R O I AI22265-01A3 awarded to R.J.G. We thank Mrs. Sally Baker for typing the manuscript. REFEP.EN(ZS
i.
2. 3. 4. 5. 6. 7. 8. 9. I0. ii.
12. 13. 14. 15 16 17 18. 19 20. 21 22. 23. 24. 25. 26. 27. 28.
Spreafico, F., and Anaclerio, A. (1977). Comp. Immunol. Series 3, 245-27 8. Fauci, A.S. (1978). J. Immunopharmacol. I, 1-25. Parrillo, J.E. and Fauci, A.S. (1979). Ann. Rev. Pharmacol. Toxicol. 19, 179-201. Grass,, R.J., Klein, T.W. and Benjamin, W.R. (1981). J. Immunopharmacol. 3, 171-192. Grass,, R.J., West, L.A., Guay, R.C., Jr. and Klein, T.W. (1982). J. Immunopharmacol. 4, 265-278. Grass,, R.J., West, L.A., Guay, R.C.,Jr. and Klein, T.W. (1983). Int. J. Immunopharmacol. 5, 267-276. Grass,, R.J. and Guay, R.C., Jr. (1983). Adv. Exptl. Med. Biol. 166, 279-28 Becker, J. and Grass,, R.J. (1985). Int. J. Immunopharmacol. 7, 839-847. Becker, J. and Grass,, R.J. (1988). Int. J. Immunopharmacol. i0, (In press). Glyglewski, R.S., Panczenko, B., Grodinska, R. and Ocetkiewicz, A. (1975). Prostaglandins i0, 343-355. Blackwell, G.J., Carnuccio, R., DiRosa, M., Flower, R.J., Langham, C.S.J., Parente, L., Persico, P., Russel-Smith, N.C. and Stone, D. (1982). Br. J. Pharmacol. 76, 185-194. Blackwell, R.J., Carnuccio, R., DiRosa, M., Flower, R.J., Parente, L. and Persico, P. (1980). Nature 287, 147-149. Hirata, F., Schiffman, E., Venkatasubramanian, K., Solomon, D. and Axelrod, J.G. (1980). Proc. Natl. Acad. Sci., U.S.A. 77, 2533-2536. DiRosa, N., Flower, R.J., Hirata, F., Parente, L. and Russo-Marie, F. (1984). Prostaglandins 28, 441-442. Cloix, J.F., Colard, 0., Rothhut, B. and Russo-Marie, F. (1983). Br. J. Pharmacol. 79, 313-321. Jardieu, P., Akasaki, M. and Ishizaka, K. (1986). Proc. Natl. Acad. Sci., U.S.A. 83, 160-164. Becker, J., Carter, S.W III and Grass,, R.J. (1986). J. Immunol. Meth. 91, i-i0. Volwerk, J.J., Pieterson, W.A., and Dehaas, G.H. (1974). Biochem. 13, 1446-1454. Irvine, R.F. (1982). Biochem. J. 204, 3-16. Gersten, D.M. and Fogler, W.F. (1979). Biochem. Pharmacol. 28, 1119-1122. Scott, W.A., Zrike, J.M., Hamill, A.L., Kempe, J. and Cohn, Z.A. (1980). J. Exp. Med. 152, 324-335. Huterer, S. and Wherrett, J. (1979). J. Lipid Res. 20, 966-973. Bonser, R.W. and Siegal, M.I. (1982). In:Phagocytosis-Past and Future (M.L. Karnovsky and L. Bolis, eds.) pp. 131-147. Academic Press, New York, NY. Hirata, F., Corcoran, B.A., Venkatasubramanian, K., Schiffman, E. and Axelrod, J. (1979). Proc. Natl. Acad. Sci., U.S.A. 76, 2640-2643. Takenawa, T., Momma, Y. and Nagai, Y. (1983). J. Immunol. 130, 2849-2855. Bonney, R.J., Wightman, P.D., Davies, P., Sadlowski, S.J., Kuehl, F.A., Jr. and Humes, J.L. (1978). Biochem. J. 176, 433-442. Shohet, S.B. (1970). J. Lab. Clin. Med. 75, 659-672. DeGeorge, J.J., Ousley, A.H., McCarthy, K.D., Morrell, P. and Lapetina, E.G. (1987). J. Biol. Chem. 262, 9979-9983.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 583-590
DEXAMETHASONE ACTION INHIBITS THE RELEASE OF ARACHIDONIC ACID FROM PHOSPHATIDYLCHOLINE DURING THE SUPPRESSION OF YEAST PHAGOCYTOSIS IN MACROPHAGE CULTURES
Jeanne L. Becker I*, Robert J. Grasso I and John S. Davis 2 Department of Medical Microbiology and Immunology I, Department of Internal Medicine 2, College of Medicine, University of South Florida Tampa, Florida, 33612 Received April 13, 1988
SUMMARY: Dexamethasone suppresses phagocytosis of heat killed Saccharomyces cerevisiae in cultures of murine peritoneal macrophages. Recent observations suggest that dexamethasone induces a phagocytic inhibitory protein that suppresses yeast ingestion by inhibiting macrophage phospholipase A2 activity. The present investigation, therefore, examined whether macrophage lipid metabolism is modulated by dexamethasone. Control and steroid treated macrophages were allowed to incorporate radiolabeled arachidonate and were incubated subsequently in the absence and presence of yeast. Following ingestion by control macrophages, arachidonate from phosphatidylcholine was readily cleaved to free fatty acid and transferred to the neutral lipid fraction. In contrast, arachidonate release was inhibited in dexamethasone treated macrophages. These results suggest that the suppression of yeast phagocytosis by dexamethasone action may be associated with the inhibition of phospholipase A2 activity. © 1988 Academic Press, Inc.
Glucocorticoid steroids are potent antiinflammatory and immunosuppressire agents that decrease host resistance to infectious diseases (1-3).
In
this regard, we have demonstrated that DEX and other glucocorticoids inhibit the ingestion of heat-killed Saccharomyces cerevisiae particles in cultures of murine resident and elicited peritoneal macrophages (4-9). The mechanism underlying this steroid induced suppressive effect on yeast phagocytosis remains to be elucidated.
In contrast to reversing
other glucocorticoid directed inhibitory responses by supplying exogenous AA (I0), phagocytic inhibitory responses in DEX-treated macrophage cultures are not reversible by supplying exogenous AA either alone or in combination with indomethacin and nordihydroguaiaretic acid (8).
More recent
*Present Address: H. Lee Moffitt Cancer Center and Research Institute, P.O. Box 280179, Tampa, FL 33682 DEX = Dexamethasone; PIP = Phagocytosis Inhibitory Protein; PLA2 = Phospholipase A2; TLC = Thin Layer Chromatography; A A = Arachidonic Acid; PA = Phosphatidic Acid; PE = Phosphatidylethanolamine; PI = Phosphatidylinositol; PC = Phosphatidylcholine; MAG = Monoacylglycerol (monopalmitoylglycerol); OH = Cholesterol; DAG = Diacylglycerol (diolein); TAG = Triacylglycerol (triolein); and BPB = p-Bromophenacyl Bromide. ABBREVIATIONS:
583
0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 153, No. 2, 1988
evidence indicates that
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DEX induces (via de nero RNA and protein synthesis)
a heat-labile, trypsin-sensitive factor termed PIP, which mediates the inhibition of yeast ingestion (9).
Since PIP activity is neutralized by
monoclonal antibodies directed against lipocortin (9), the antiphagocytic factor appears to belong to this family of PLA2 inhibitory proteins that are induced by glucocorticoids
(11-16).
Therefore, we postulate that glucocorticoid action inhibits PLA2 activity which, in some fashion, interferes with the normal functioning of macrophage membranes required for yeast phagocytosis to occur.
Although AA
itself, as well as its eicosanoid products, may not directly influence the DEX induced inhibition of phagocytosis, this suppressive response may be related to altered lipid metabolism. Our results suggest that PLA2 activity is suppressed
during the inhibition of phagocytosis in DEX treated macro-
phage cultures.
MATERIALS AND METHODS
Macrophage Cultures Cultures of resident peritoneal macrophages were established from leukocytes lavaged from male C57BL/6 mice as described in detail elsewhere (17). When the cultures were established, control macrophages were supplied culture medium (Dulbecco's Modified Eagle Medium supplemented with antibiotics and 10% heat-inactivated fetal bovine serum) lacking the steroid and the experimental macrophages received medium supplemented with i ~M DEX. For the yeast ph~gocytosis assays, each well of a 96 well microtiter plate contained 1.5xl0 adherent macrophages in 0.15 ml of medium. ~or the lipid studies, each well of a 24 well cluster plate contained 1.3x10 v adherent macrophages in 1.5 ml of mediumbC All control and steroid treated cultures were incubated for 3 days at 37 in an atmosphere of 95% air-5% CO 2 prior to initiating the experiments. Yeast Phagocytosis Assays Ingestion of [3H] S. cerevisiae by macrophages in the wells of the microtiter plates was ~ a s u r e d w~th a radiometric yeast phagocytosis assay (17). Phagocytic capacities of macrophages in replicate control and i ~M DEX treated cultures are defined as the mean CPM values + S.E.M. Radiolabeling and Extraction of Macrophage Lipids Three day old control and I}IM DEX t r ~ t e d macrophages were exposed for 4 hr at 37 °C to either i ~Ci/ml of [I-~C]-AA (spec. act. = 58 mCi/mmol) or 6.7 ~Ci/ml of [5,6,8,9,11,12,14, 15-3H] -AA (spec. act. = 220 mCi/mmol; Amershsm, Inc., Arlington Heights, IL). After removing unincorporated radioactivity, the cultures were supplied respective fresh media and incubated at 37 °C for an additional 15 min in the absence and presence of non-radioactive heat-killed S. cerevisiae. All media was removed after placing the cultures at 0°C,-Macropnages were scraped from each well into 3 ml of absolute methanol at 0°C, to which 6 ml of chloroform and 0.25 ml of 1 N HCI was added. The ratio of chloroform/methanol/HCl was 2/1/0.25. The solution was vortexed for 30 sec and centrifuged for i0 min at i000 x g at 4°C. The aqueous phase was discarded. This washing procedure was repeated sequentially with chloroform/methanol/0.1 N HCI followed by chloroform/methanol/0.01 N HCI (3/48/47 v/v). The organic phase containing the extracted lipids was stored overnight at -70°C. On the following day, the extracts
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were placed at 0°C, centrifuged as described above, and any remaining aqueous phase was discarded. The extracts were dried under a stream of nitrogen and dissolved in chloroform/methanol/water at 75/25/2 (v/v). TLC of Macrophage Phospholipids Two dimensional Redi-Coat silica gel TLC plates containing 10% magnesium acetate (Supelco, Bellefonte, PA) were developed to within i cm of the top in methanol/water (2/3) containing 1% potassium oxalate. Thereafter, the plates were air dried and activated at ll0°C for 30 min. The TLC plates were developed in chloroform/acetone/methanol/acetic acid/water (40/15/13/12/8 v/v) to within 2 cm from their upper edges. The plates were air dried and the lipids were identified by exposure to iodine vapors. Sample lipids were compared to standards obtained from Sigma Chemical Co. (St. Louis, MO). The material was scraped from the plate and radioactivity in each band was measured by liquid scintillation spectrometry, TLC of Macrophage Neutral Lipids The TLC plates were activated at II0UC for 45 min and spotted with the macrophage lipid extracts. The TLC plates were first developed in ether/benzene/ethanol/acetic acid (40/50/2/2 v/v) to 5 cm from the top. The plates were then air dried and developed further in ether/hexane (6/94 v/v) to within 2 cm from the upper edges. After the plates were air dried, the neutral lipid containing bands were visualized, scored, collected by scraping, and radioactivity measured. Authentic neutral lipid standards were obtained from Sigma Chemical Co. RESULTS
Suppression of Yeast Ingestion by BPB Our hypothesis predicts that inhibiting PLA2 activity should inhibit yeast phagocytosis.
Hence, we measured the phagocytic capacities of
macrophages exposed to BPB, an inhibitor of PLA2 (18).
Dose response
studies revealed that i0 pM BPB suppressed yeast ingestion but was not toxic to the macrophages as determined by trypan blue dye exclusion tests. Accordingly, 3 day old control and i pM DEX treated macrophage cultures were incubated at 37°C for up to 45 min in the absence and presence of i0 ~MBPB.
Yeast phagocytosis assays, each lasting 15 min, were initiated at
the time the phospholipase inhibitor was supplied, and at 15 min intervals thereafter.
Figure I illustrates that BPB suppressed yeast ingestion
almost completely in control cultures after 45 min.
The steroid alone
elicited a 55% inhibitory response after 3 days and 15 min of treatment with BPB almost completed suppressed yeast in the DEX treated cultures. The initial rates of the inhibitory responses elicited by BPB were nearly parallel in the control and steroid treated cultures.
Separate experiments
with the phospholipase inhibitors, quinacrine (i0 ~/~) and tetracaine (i00 pM), yielded similar results (data not shown). Modulation of Macrophage Lipid Metabolism Since phospholipid turnover can be measured by changes in AA (19), we examined the distribution of radiolabeled AA among phospholipids and
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- c
11
4\
o CONTROL
_Lx
10 9 8
~T " I ~
\
? ,.p.o 7 m
0.
01#M DEX A IO~M BPB • 1 0 ~ BPB+ 1.M DEX [] BACKGROUND
k
6
u
3 2 1
0
15
30
45
TIME OF EXPOSURE TO BPB Iminl FIGURE 1.
Time Course o f BPB I n h i b i t i o n
o f Yeast P h a g o c y t o s i s i n C o n t r o l
Dan - - - ~ T r e a t e d Macrophage Cultures. Murine resident peritoneal macrophages were incubated for 3 days in the absence and presence of 1 NM DEX. On day 3, the cultures were exposed to i0 ~M BPB for up to 45 min. Phagocytic capacities were measured over 15 min assay periods in both sets of cultures at the times indicated by the symbols, which represent mean CPM values + S.E.M. (N=3).
neutral lipids in 3 day old control and I NM DEX treated macrophages after 15 min incubation periods in the absence and presence of unlabeled yeast particles. Table 1 shows the distribution of radioactivity incorporated into phospholipids and neutral lipids.
Total CPM incorporated into lipids of
individual samples was variable.
In these studies, 8,074 + 3,122 CPM were
present in phospholipids in nonphagocytizing control cultures with 2,464 + 1,415 CPM in neutral lipids.
In noningesting DEX treated cultures, 4,457 +
965 CPM were present in phospholipids and 1,259 + 279 CPM in neutral lipids, thus suggesting that basal phospholipid [urnover was reduced in DEX treated macrophages.
Following yeast phagocytosis, control cultures
exhibited 15,051 + 4,525 CPM in phospholipids and 19,604 + 11,402 CPM in neutral lipids; DEX treated cultures had 9,728 + 4,984 CPM in phospholipids and 4,933 + 2,985 CPM in neutral lipids f o l l o w J g yeast ingestion. Phosp~olipids contained 82% of the total radioactivity in control macrophages not exposed to yeast with over 50% of the isotope found in PC. The extracted neutral lipid fraction contained 18% of the radioactivity in
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TABLE
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Dexamethasone Action Inhibits the Conversion of [14C] AA from PC to Neutral Lipids During Yeast Phagocytosis [14C] AA-labeled Lipids a
Macrophage Cultures
15 Min Exposure to Unlabeled Yeast
Phospholipids (% of Total) Neutral PC
PE+PA
PI
Otherb
Lipids c
Control
-
55 + 2
16 + 2
7 + i
4 + I
18 + 5
Control
+
27 + 4
16 + 2
3 + 1
5 + 1
49 + 9
i ~M D E X
-
47 + 3
19 + 3
5 + 2
7 + 1
22 + 1
i pM DEX
+
40 + 3
17 + 5
4 + I
9 + I
29 + 6
a b c
Mean Percentage + S.E.M. (N=3), based on total radioactivity of sample. This fraction contained PI mono-and diphosphates. See Table 2 for individual neutral lipids that were measured in separate experiments.
these noningesting macrophages.
When phagocytosis was stimulated by the
addition of yeast, the percentage of radioactivity in the PC fraction decreased by 28% and in the neutral lipid fraction increased by 31%.
In
contrast, when DEX treated cultures were exposed to yeast, only a 7% decrease in radioactivity in PC occurred, with a corresponding increase of 7% in the neutral lipids. On a percentage basis, no major changes in radioactivity were observed in the remaining phospholipid fractions in either control or steroid treated cultures, regardless of whether yeast was present or not. Table 2 reveals the results of separate experiments that show the distribution of radioactivity in the major neutral lipids in macrophage cultures.
In the absence of phagocytosis, the total radioactivity in
neutral lipids in control cultures was 36,926 + 10,652 CPM; DEX treated cultures contained 4,750 + 1,348 CPM in neutral lipids under this condition.
Following yeast ingestion, 36,994 + 17,585 CPM were present in
neutral lipids in control cultures and 21,486 + 3,643 CPM were present in neutral lipid extracted from steroid treated macrophages. to control macrophages in the absence of yeast,
Thus, relative
the steroid treated
macrophages incorporated only 13% as much [3H]-AA into neutral lipids. During yeast phagocytosis in control cultures, 19% increase was observed in
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TABLE 2. Distribution of [3H] AA in Neutral Lipids of Control and Glucocorticoid Treated Macrophages in the Absence and Presence of Yeast Phagocytosis [3H] AA-labeled Neutral Lipids (% of Total) a Macrophage
15 Min
Cultures
Exposure to Unlabeled Yeast
AA
TAG
DAG
CE
MAG
CH
Control
-
9 +
2
74 +
3
6 + I
8 + I
3 + i
1 + 0
Control
+
28 +
2
54 +
3
8 + 2
4 + 0
5 + 1
2 + i
i ~M DEX
-
23 .+ 12
. 52 + II .
9. + 0
6 + I
I + 0
1 ~M DEX
+
15 +
3 + I
I + 0
a
2
69 +
4
. 9 + i.
8 + I
5 + I
Mean Percentage + S.E.M. (N=3), based on total radioactivity of sample.
the portion of radioactivity associated with free AA. This effect was accompanied by a 20% decrease in the percentage of radioactivity associated with TAG.
In contrast, when DEX treated macrophages were exposed to yeast,
a minimal 8% decrease occurred in the percentage of radiolabeled free AA. This effect was accompanied by a 17% increase in the percentage of radioactive TAG.
No major changes occurred in the percentage of
radioactivity associated with DAG, CE, MAG and CH in either control or steroid treated macrophages,
regardless of whether they were exposed to
yeast.
DISCUSSION These results demonstrate that glucocorticoid action inhibits phagocytosis potentially by modulating lipid metabolism. cultures,
Relative to control
the turnover of PC is suppressed in DEX treated macrophages
exposed to yeast.
Although DEX treated macrophages
number of yeast particles
ingest only a limited
(4-6), higher CPM values were recovered when the
cells were allowed to ingest yeast; similar results also occurred in control cultures.
This finding may indicate that the radiolabeled AA, which
might otherwise he released into the culture fluids,
is readily reesterified
into cellular lipid during the process of phagocytosis.
However, the total
CPM present in DEX treated cultures was about 50% of control values and the net increase in phospholipid turnover induced by phagocytosis was much greater in control cultures.
Moreover,
the relative amounts of the radio-
labeled arachidonate associated with each of the lipid fractions changes very little in macrophages exposed to DEX, regardless of whether or not yeast is supplied to the treated cultures.
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Based upon our previous studies, we postulate that the alterations of lipid metabolism produced by DEX may be mediated by steroid induced PIP. The data collected on the radiolabeled lipids presented in Table i implicate PLA2 as a likely target for DEX action.
This interpretation is supported
by the results of the BPB kinetic experiments, as well as by experiments with the phospholipase inhibitors, quinacrine and tetracaine. others have
Furthermore,
demonstrated that tetracaine suppresses phagocytosis of sheep
erythrocytes by murine elicited macrophages (20). In cultures of non-steroid treated, non-phagocytizing macrophages,
the
distribution of AA among the various lipid fractions agrees very well with previous studies performed by others with murine peritoneal macrophages (19,21) and with rabbit alveolar macrophages (22).
A similar distribution
of AA in phospholipids is present in DEX treated macrophages that are not exposed to yeast, even though the total amounts of radioactivity associated with phospholipids are considerably reduced relative to the controls. The release of AA from PC during yeast phagocytosis by control macrophages reflects normally functioning membrane activity required for ingestion.
Studies with HL60 lymphoma cells have shown that a shift in AA from
PC to neutral lipids also occurs when these cells are stimulated with either the chemotactic peptide N-formyl Nle-Leu-Phe-Nle-Tyr-Lys or the calcium ionophore A23187 (23), both of which activate PLA2 (24,25).
In
addition, stimulation of murine peritoneal macrophages with zymosan results in the release of AA from PC with a corresponding increase of the fatty acid in neutral lipids (26).
Thus, exposing control macrophage cell mem-
branes to an external physical stimulus, such as yeast particles, may result in increased PLA2 activity accompanied by the release of the isotope from PC to AA. We also observed that control macrophages exhibit a decreased percentage of AA in TAG and corresponding increase in free AA following yeast ingestion.
These changes in arachidonate distribution in the neutral
lipids may be associated with the activation of TAG lipase, whose activity may be enhanced during phagocytosis (27).
In contrast, DEX treated macro-
phages exhibit an increased percentage of AA in TAG, which may reflect decreased levels of the activity of this enzyme. Taken together, the evidence presented in this report supports the hypothesis that glucocorticoid action inhibits phagocytosis by suppressing PLA2 activity, presumably via PIP activity.
This notion is strengthened by
the recent finding that glucocorticoids preferentially inhibit PLA2 within C62B glioma cells in vivo, but exert very little inhibitory effect on phospholipase C (28).
Whether this in fact also occurs in DEX treated
macrophages remains to be investigated.
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ACKNOWLEDGEMENTS
This research was supported in part by USPHS NIH Grant Number 1 R O I AI22265-01A3 awarded to R.J.G. We thank Mrs. Sally Baker for typing the manuscript. REFEP.EN(ZS
i.
2. 3. 4. 5. 6. 7. 8. 9. I0. ii.
12. 13. 14. 15 16 17 18. 19 20. 21 22. 23. 24. 25. 26. 27. 28.
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