- Email: [email protected]
Synthesis of prostaglandins by subpopulations of human peripheral blood monocytes
PROSTAGLANDINS SYNTHESIS OF PROSTAGLANDINS BY SUBPOPULATIONS OF IWMAN PERIPHERAL BLOOD MONOCYTES
M.E. Goldyne and J.D. Stobo
From the Department of Dermatology and Section of Rheumatology/ Clinical Immunology, Department of Medicine, Moffitt Hospital, University of California, 94143.
ABSTRACT
The ability of monocytes/macrophages to regulate various aspects of immunologic responses may in part depend on their release of soluble substances such as prostaglandins. Using quantitative gas-liquid chromatography/mass spectrometry, prostaglandin E2 was found to be the major prostaglandin synthesized in culture by human peripheral blood monocytes. Subjecting these cells to discontinuous density gradient fractionation demonstrated significant differences in the synthesis of prostaglandins E2 and El among the resulting monocyte subpopulations.
INTRODUCTION
The E prostaglandins appear capable of modulating a variety of physiologic functions including those of an immunologic nature (1,2). Their synthesis by immunologic regulatory cells could conceivably provide one means of achieving control of immunologic effector cell function. This concept is supported by studies in both animals and humans demonstrating that E prostaglandins are produced by monocyteslmacrophages and in turn have the capacity to inhibit a series of T celldependent responses in vitro (3-6). Moreover, it has been postulated that excessive production of prostaglandin (PG) E2 by human monocytesf macrophages may be causally related to the in vivo T cell hypofunction present in some patients with Hodgkin's disease (7).
Studies recently performed in our laboratory suggest that certain non-prostaglandin-relatedimmunoregulatory functions of monocytesf
NOVEMBER
1979 VOL. 18 NO. 5
687
PROSTAGLANDINS macrophages reflect the capabilities of subpopulations of these cells rather than a constitutive function of the entire cell line (8,9). Therefore, in the context of PG synthesis, we decided to examine whether or not subpopulations of human peripheral blood monocytes (M$), obtained by density gradient fractionation, differed in their capacities to synthesize prostaglandins.
MATERIATS AND METHODS
Isolation and Culture of Human Peripheral Blood M8
Human peripheral blood Ma were isolated according to the methods recently published by Raff et al (8). The purified cells (95% esterase posittie, 90% phagocytic) were platelet depleted by washing twice in calcium-, magnesium-free phosphate buffered saline containing 0.5 mM EDTA followed by centrifugation through 7.5% bovine serum albumin (BSA). The cells were then fractionated into five subpopulations by sedimentation through a discontinuous density gradient consisting of 1 ml fractions of 17, 19, 21, 23 and 25% BSA as previously described (8). Fractions 1 (17/19% interface, > 95% Ma, > 95% viability), 2 (19/21% interface, > 95% M@, > 95% viability), 3 (21/23% interface > 95% M@, > 95% viability), and 4 + 5 (23125% interface + pellet, > 75% M(b,> 70% viability) were individually incubated at a concentration of 1 x lo6 cells/ml. Fractions 4 + 5 were combined due to the low number of cells sedimenting to these regions of the gradient. Cell viability was determined by trypan blue exclusion.
Thin-layer Chromatography 14 To study the synthesis of [14Cl PGs from [ Cl arachidonic acid (A.A.) by the Mfl subpopulations, the individual fractions were incubated with 1.3 mg of [l-14C] arachidonic acid (500,000 dpm) in 1.5 ml of tris buffer, pH 7.4 at 37'C for thirty minutes in a shaking bath.
Following
the incubation the lipids were extracted three times with 0.5 volumes of diethyl ether after acidifying (pH 3) the supernatants with O.lM HCl. The ether extracts were evaporated and the residues redisolved in ethyl
688
NOVEMBER
1979 VOL. 18 NO. 5
PROSTAGLANDINS
acetate and applied to glass plates coated with silica gel G.
The
plates were developed once or twice (to separate thromboxane (TX)B2 from PGE2) in a solvent system consisting of the organic phase of ethyl acetate: iso-octane: acetic acid: water (9:5:2:10) following an initial 2h phase equillibration. The radioactive peaks were located using a Berthold LB 2760 TLC scanner. Co-chromatographed standard PGE2, 6 ketoPGFla, PGF20, PGD2 and TXB2 were visualized using 10% phosphomolybdic acid spray.
Selective Ion Monitoring
PGE2 and PGEl were quantitated using gas-liquid chromatography/mass spectrometry (gc/ms) in the selective ion monitoring mode according to the methods of Axen et al (10) and Goldyne and Hammarstrom (11). One ng of [3,3,4,4-2H4]PGE2, 1 ug of [3,3,4,4,5,6-2HI PGEl and 100,000 dpm 4 each of 13H] PGE2 (sp. act 160 Ci/m mol) and [ HI PGEl (59 Ci/mmol) were added (as internal standards and tracers respectively) to supematants from the individual M8 fractions incubated for 48 h in EPMI 1640 medium containing either 10% fetal calf, or autologous serum (the source of serum did not alter relative or absolute levels of PGE synthesized). The total supernatant from each incubation (1 ml) was cleared of protein by precipitation with 3 volumes of absolute ethanol followed by filtration of the supernatant. Following vacuum evaporation of the ethanol, the remaining aqueous medium was expanded to 1 ml, acidified to pH 3 with 0.1 M HCl, and extracted 3 times with 0.5 volume of diethyl ether. The combined ether phases were evaporated and the remaining residue dissolved in 5 ml of hexane and applied to a 250 mg silicic acid column. The column was eluted with 5 ml lo%, 7.5 ml 20% diethyl ether in hexane followed by 15 ml of ethyl acetate which eluted the PGEs.
Following
evaporation of the ethyl acetate, the PGEs were methylated with ethereal dizaomethane (12). The resulting methyl esters were subjected to thinlayer argentation chromatography followed by further purification and derivatization to 0-methyloxime-acetatesas previously described (10,ll). For PGE2, the intensities of the ions at m/e 419 (non-deuterium labeled) and 423 (deuterium labeled; m - 60) were monitored against time. For PGEl, the ions at m/e 330 and 335 (M - (120 + 31)) were monitored. An
NOVEMBER
1979 VOL. 18 NO. 5
689
PROSTAGLANDINS
LRB 9000 gas chromatograph/mass spectrometer equipped with an accelerating voltage alternator and a 1.5 m column of 1% OV-1 on 60/80 mesh Supelcoport was used for the analyses. Operating conditions were: electron energy = 25 eV, trap current = 60 uA, column temp. = 235'C, helium gas flow = 20 mllmin.
RESULTS AND DISCUSSION
Figure 1 shows the thin-layer radiochromatogramsof the lipid extracts obtained following incubation of the M(ii subpopulationswith [L4C] A . A .
In agreement with the recent paper by Morley et al., (13),
radioactive peaks with Rf values coincident with standard PGE2 and TXB2 could be identified. There was no activity corresponding to 6 ketoPGFla, PGF2a, or PGD2.
Using single development of the TLC plates also
showed peaks corresponding to HETE and HHT (data not shown). The TLC profiles suggested that the subpopulationsmight vary in their synthesis of PGE2 as well as TXB2.
Because E prostaglandins have been directly
associated with immunoregulatory function, we decided to first examine the quantitative profiles of PGEl and PGE2 synthesis by the M@ fractions.
690
NOVEMBER
1979 VOL. 18 NO. 5
PROSTAGLANDINS
Thin-layer radiochromatograms of lipid extracts from human
Figure 1:
peripheral blood M@ fractions following 30 minute incubations with Circles represent positions of co-chromatographed standards
[14C1 A.A.
as labeled. Final recovery of racioactivity in each fraction was: 76%, 2 = 55%, 3 = 59%, 4 + 5 = 56%.
I=
See text for details.
Figure 2 shows the quantitative profiles of PGE2 synthesis as determined by the selective ion monitoring assays. PGEl could only be detected in culture fluid from cells sedimenting in fraction 3 (2.3 ng/106 cells - data not shown) and this level was only l/16 that of PGE2 (36.7 ng/106 cells) synthesized by the same cells. Therefore, PGE2 is the major prostaglandin produced by the fractionated M@.
50
40
30
20
10 / 1
DENSITY
Figure 2:
2
3
GRADIENT
4+5
FRACTION
Levels of PGE2 accumulating in the culture fluids from 48 h
incubations of gradient fractionated PBM.
Mean values (n=2) + S.E.M.
are expressed as ng PGE2 per lo6 cells. See text for statistical analysis.
Statistically (one-tailed Student's t test), no difference could be documented between the levels of PGE2 produced by the cells in fractions 2 or 3.
However, a significant difference was found between the levels
in fractions 2 or 3 and the levels produced by fraction 1 (p < 0.025) or
NOVEMBER
1979 VOL. 18 NO. 5
691
PROSTAGLANDINS fraction 4 -t 5 (p < 0.05). The levels of PGE2 produced by cells in fraction 1 were not statistically greater than those produced by fractions 4 + 5 when corrections were made for cell viability.
Whether or not the significant variations in PGE2 levels found among M@ fractions after 48 h culture are the result of differences in rates of synthesis or degradation is not directly demonstrated by these studies. However, after 30 minute incubations of the different M&?J fractions with [14Cl A.A. (figure l), different peak heights for PGE2 (as well as TXB2) are apparent. Furthermore, incubating the fractionated cells with 13~l-p~~2 for 30 minutes (the same incubation time used for the [14C] A.A. conversion studies) failed to show any detectable breakdown of the PGE2 (data not shown). These observations would suggest that differing activities of PG endoperoxide synthase and/or PG endoperoxide E isomerase, rather than differential catabolism of PGE2, account for the variations in PGE2 synthesis among the M8.
The differences in PGE2 synthesis among the M8 subpopulations could also be postulated to result from the differential activation of the cells by the fractionation procedure itself. By measuring the specific activities of the enzymes (5'-nucleotidaseand acid phosphatase) claimed to represent biochemical correlates of monocyte activation (14-16), fraction 1 cells were found to be more activated (i.e. lower 5'-nucleotidase levels, higher acid phosphatase levels) than cells sedimenting in fractions 3 or 4 + 5 (17). Therefore, no simple correlation was observed between the levels of PGE2 among the fractionated M# and their degree of activation as determined by enzyme analyses. Furthermore, when fraction 1 cells were incubated in concentrations of BSA comparable to those present in high density fractions (25%) and vice versa for 45 minutes (4'C) before carrying out the 48 h
cultures, there was no alteration
in their relative synthesis of PGE2.
These results appear to exclude
density gradient-related activation of the subpopulations as an explanation for the differential synthesis of PGE2.
The relatively low levels of PGE2 generated by fraction 4 + 5 could conceivably be attri"qltedto the lower viability and % M@ in this
692
NOVEMBER
1979 VOL. 18 NO. 5
PROSTAGLANDINS
fraction when compared to the other fractions. However, correcting for viability and % ML? in fraction 4 + 5 still gave levels of PGE2 (3.34 + 1.48 ng/106 cells) statistically lower than those in fraction 2 or 3 (p < 0.05). Therefore, cell viability does not account for the different levels of PGE2
produced by the MQifractions.
In summary, the data presented in this report document significant differences among human peripheral blood M@ subpopulationswith regard to the synthesis of PGE2 and PGEl.
If PGE2 synthesis represents a means
through which M@ can exert an immunoregulatory influence, our data support the existance of heterogeneity among these cells in their capacity to effect such PGE2-mediated regulation. It could be postulated that pathologically induced alterations in the relative numbers of the individual M@ subpopulations might lead to serious alterations in PGE2mediated immunoregulation. In addition, the profiles of TKA2 synthesis (as measured by TKB2) may reveal further heterogeneity among the human peripheral blood M@ fractions in regard to arachidonic acid metabolism. These possibilities are currently being explored.
ACKNOWLEDGEMENTS
We wish to thank Ms. M. Newton, Ms. K. Chinn and Mr. G. Benthin for their technical assistance. Drs. U. Axen and J. Pike of the Upjohn Co. (Kalamazoo, Mich.) kindly supplied the deuterium-labeled and standard prostaglandins. M.E.G. is the recipient of a Clinical Investigator Award from the National Institute of Arthritis, Metabolism, and Digestive Diseases. J.D.S. is an Investigator of the Howard Hughes Medical Institute. This work was supported by USPHS Grants AI-14014 and AI14572.
NOVEMBER 1979VOL.18NO.S
693
PROSTAGLANDINS
REFERENCES
1.
Pelus, L. and Strausser, H. Prostaglandins and the immune response. Life Sciences 20:903-913, 1977.
2.
Goldyne, M.E. Prostaglandins and the modulation of immunologic responses. Int. J. Dermatol. 16:701-712, 1977.
3.
Kurland, J.I. and Bockman, R.J. Prostaglandin E production by human blood monocytes and mouse peritoneal macrophages. J. Exp. Med. 147:952-957, 1978.
4.
Stockman, G.D. and Mumford, D.M. The effect of prostaglandins on the in vitro blastogenic response of human peripheral blood lymphocytes. Exp. Hematol. 2~65-72, 1974.
5.
Lichtenstein, L.M., Gillespie, E., Bourne, HR. and Henney, C.S. The effects of a series of prostaglandins on in vitro models of the allergic response and cellular immunity. Prostaglandins 2:519528, 1972.
6.
Bray, M.A., Gordon, D. and Morley, J. Regulation of lymphokine secretion by prostaglandins. Agents Actions 6:171-175, 1976.
7.
Goodwin, J.S., Messner, R.P., Bankhurst, A.D., Peake, G.T., Saiki, J.H. and Williams, R.C. Prostaglandin producing suppressor cells in Hodgkin's disease. N. Engl. J. Med. 297:963-968, 1978.
8.
Raff, H.V., Cochrum, K. and Stobo, J.D. Macrophage-T cell interactions in the Con A induction of human suppressor T cells. J. Immunol. 121:2311-2315, 1978.
9.
Raff, H.V. and Stobo, J.D.
Unpublished results.
10. Axen, U., Green, K., Horlin, D. and Samuelsson, B. Mass spectrometric determination of picomole amounts of prostaglandin E and using synthetic deuterium-labeled carriers. Biochem. B zophys. & . comm . 45:519-529, 1971. 11. Goldyne, M.E. and Hammarstrom, S. Quantitative determination of prostaglandins El and Fla by multiple ion analysis. Anal. Biochem. 88:675-681, 1978. 12.
Schlenk, H., Gellerman, J.L. Esterification of fatty acids with diazomethane on a small scale. Anal. Chem. 32(11): 1412-1414, 1960.
13. Morley, J., Bray, M.A., Jones, R.W., Nugteren, D.H. and Van Drop, D.A. Prostaglandin and thromboxane production by human and guinea pig macrophages and leukocytes. Prostaglandins 17:729-736, 1979.
694
NOVEMBER
1979 VOL. 18 NO. 5
PROSTAGLANDINS
14. North, R.J. The concept of the activated macrophage. J. 121:806-809, 1978.
Immunol.
15. Karnovsky, M.I. and Lazdins, J.K. Biochemical criteria for activated macrophages. J. Immunol. 121:809-813, 1978. 16. Cohn, Z.J. The activation of mononuclear phagocytes: Fact, fancy and future. J. Immunol. 121:813-816, 1978. 17. Picker, L., Raff, H.V., Goldyne, M.E. and Stobo, J.D. Biochemical heterogeneity among human monocytes. Submitted for publication. (Received 4/6/1979 Accepted 10/15/1079) Editor: Shozo Yamamoto
NOVEMBER
1979 VOL. 18 NO. 5
695
M.E. Goldyne and J.D. Stobo
From the Department of Dermatology and Section of Rheumatology/ Clinical Immunology, Department of Medicine, Moffitt Hospital, University of California, 94143.
ABSTRACT
The ability of monocytes/macrophages to regulate various aspects of immunologic responses may in part depend on their release of soluble substances such as prostaglandins. Using quantitative gas-liquid chromatography/mass spectrometry, prostaglandin E2 was found to be the major prostaglandin synthesized in culture by human peripheral blood monocytes. Subjecting these cells to discontinuous density gradient fractionation demonstrated significant differences in the synthesis of prostaglandins E2 and El among the resulting monocyte subpopulations.
INTRODUCTION
The E prostaglandins appear capable of modulating a variety of physiologic functions including those of an immunologic nature (1,2). Their synthesis by immunologic regulatory cells could conceivably provide one means of achieving control of immunologic effector cell function. This concept is supported by studies in both animals and humans demonstrating that E prostaglandins are produced by monocyteslmacrophages and in turn have the capacity to inhibit a series of T celldependent responses in vitro (3-6). Moreover, it has been postulated that excessive production of prostaglandin (PG) E2 by human monocytesf macrophages may be causally related to the in vivo T cell hypofunction present in some patients with Hodgkin's disease (7).
Studies recently performed in our laboratory suggest that certain non-prostaglandin-relatedimmunoregulatory functions of monocytesf
NOVEMBER
1979 VOL. 18 NO. 5
687
PROSTAGLANDINS macrophages reflect the capabilities of subpopulations of these cells rather than a constitutive function of the entire cell line (8,9). Therefore, in the context of PG synthesis, we decided to examine whether or not subpopulations of human peripheral blood monocytes (M$), obtained by density gradient fractionation, differed in their capacities to synthesize prostaglandins.
MATERIATS AND METHODS
Isolation and Culture of Human Peripheral Blood M8
Human peripheral blood Ma were isolated according to the methods recently published by Raff et al (8). The purified cells (95% esterase posittie, 90% phagocytic) were platelet depleted by washing twice in calcium-, magnesium-free phosphate buffered saline containing 0.5 mM EDTA followed by centrifugation through 7.5% bovine serum albumin (BSA). The cells were then fractionated into five subpopulations by sedimentation through a discontinuous density gradient consisting of 1 ml fractions of 17, 19, 21, 23 and 25% BSA as previously described (8). Fractions 1 (17/19% interface, > 95% Ma, > 95% viability), 2 (19/21% interface, > 95% M@, > 95% viability), 3 (21/23% interface > 95% M@, > 95% viability), and 4 + 5 (23125% interface + pellet, > 75% M(b,> 70% viability) were individually incubated at a concentration of 1 x lo6 cells/ml. Fractions 4 + 5 were combined due to the low number of cells sedimenting to these regions of the gradient. Cell viability was determined by trypan blue exclusion.
Thin-layer Chromatography 14 To study the synthesis of [14Cl PGs from [ Cl arachidonic acid (A.A.) by the Mfl subpopulations, the individual fractions were incubated with 1.3 mg of [l-14C] arachidonic acid (500,000 dpm) in 1.5 ml of tris buffer, pH 7.4 at 37'C for thirty minutes in a shaking bath.
Following
the incubation the lipids were extracted three times with 0.5 volumes of diethyl ether after acidifying (pH 3) the supernatants with O.lM HCl. The ether extracts were evaporated and the residues redisolved in ethyl
688
NOVEMBER
1979 VOL. 18 NO. 5
PROSTAGLANDINS
acetate and applied to glass plates coated with silica gel G.
The
plates were developed once or twice (to separate thromboxane (TX)B2 from PGE2) in a solvent system consisting of the organic phase of ethyl acetate: iso-octane: acetic acid: water (9:5:2:10) following an initial 2h phase equillibration. The radioactive peaks were located using a Berthold LB 2760 TLC scanner. Co-chromatographed standard PGE2, 6 ketoPGFla, PGF20, PGD2 and TXB2 were visualized using 10% phosphomolybdic acid spray.
Selective Ion Monitoring
PGE2 and PGEl were quantitated using gas-liquid chromatography/mass spectrometry (gc/ms) in the selective ion monitoring mode according to the methods of Axen et al (10) and Goldyne and Hammarstrom (11). One ng of [3,3,4,4-2H4]PGE2, 1 ug of [3,3,4,4,5,6-2HI PGEl and 100,000 dpm 4 each of 13H] PGE2 (sp. act 160 Ci/m mol) and [ HI PGEl (59 Ci/mmol) were added (as internal standards and tracers respectively) to supematants from the individual M8 fractions incubated for 48 h in EPMI 1640 medium containing either 10% fetal calf, or autologous serum (the source of serum did not alter relative or absolute levels of PGE synthesized). The total supernatant from each incubation (1 ml) was cleared of protein by precipitation with 3 volumes of absolute ethanol followed by filtration of the supernatant. Following vacuum evaporation of the ethanol, the remaining aqueous medium was expanded to 1 ml, acidified to pH 3 with 0.1 M HCl, and extracted 3 times with 0.5 volume of diethyl ether. The combined ether phases were evaporated and the remaining residue dissolved in 5 ml of hexane and applied to a 250 mg silicic acid column. The column was eluted with 5 ml lo%, 7.5 ml 20% diethyl ether in hexane followed by 15 ml of ethyl acetate which eluted the PGEs.
Following
evaporation of the ethyl acetate, the PGEs were methylated with ethereal dizaomethane (12). The resulting methyl esters were subjected to thinlayer argentation chromatography followed by further purification and derivatization to 0-methyloxime-acetatesas previously described (10,ll). For PGE2, the intensities of the ions at m/e 419 (non-deuterium labeled) and 423 (deuterium labeled; m - 60) were monitored against time. For PGEl, the ions at m/e 330 and 335 (M - (120 + 31)) were monitored. An
NOVEMBER
1979 VOL. 18 NO. 5
689
PROSTAGLANDINS
LRB 9000 gas chromatograph/mass spectrometer equipped with an accelerating voltage alternator and a 1.5 m column of 1% OV-1 on 60/80 mesh Supelcoport was used for the analyses. Operating conditions were: electron energy = 25 eV, trap current = 60 uA, column temp. = 235'C, helium gas flow = 20 mllmin.
RESULTS AND DISCUSSION
Figure 1 shows the thin-layer radiochromatogramsof the lipid extracts obtained following incubation of the M(ii subpopulationswith [L4C] A . A .
In agreement with the recent paper by Morley et al., (13),
radioactive peaks with Rf values coincident with standard PGE2 and TXB2 could be identified. There was no activity corresponding to 6 ketoPGFla, PGF2a, or PGD2.
Using single development of the TLC plates also
showed peaks corresponding to HETE and HHT (data not shown). The TLC profiles suggested that the subpopulationsmight vary in their synthesis of PGE2 as well as TXB2.
Because E prostaglandins have been directly
associated with immunoregulatory function, we decided to first examine the quantitative profiles of PGEl and PGE2 synthesis by the M@ fractions.
690
NOVEMBER
1979 VOL. 18 NO. 5
PROSTAGLANDINS
Thin-layer radiochromatograms of lipid extracts from human
Figure 1:
peripheral blood M@ fractions following 30 minute incubations with Circles represent positions of co-chromatographed standards
[14C1 A.A.
as labeled. Final recovery of racioactivity in each fraction was: 76%, 2 = 55%, 3 = 59%, 4 + 5 = 56%.
I=
See text for details.
Figure 2 shows the quantitative profiles of PGE2 synthesis as determined by the selective ion monitoring assays. PGEl could only be detected in culture fluid from cells sedimenting in fraction 3 (2.3 ng/106 cells - data not shown) and this level was only l/16 that of PGE2 (36.7 ng/106 cells) synthesized by the same cells. Therefore, PGE2 is the major prostaglandin produced by the fractionated M@.
50
40
30
20
10 / 1
DENSITY
Figure 2:
2
3
GRADIENT
4+5
FRACTION
Levels of PGE2 accumulating in the culture fluids from 48 h
incubations of gradient fractionated PBM.
Mean values (n=2) + S.E.M.
are expressed as ng PGE2 per lo6 cells. See text for statistical analysis.
Statistically (one-tailed Student's t test), no difference could be documented between the levels of PGE2 produced by the cells in fractions 2 or 3.
However, a significant difference was found between the levels
in fractions 2 or 3 and the levels produced by fraction 1 (p < 0.025) or
NOVEMBER
1979 VOL. 18 NO. 5
691
PROSTAGLANDINS fraction 4 -t 5 (p < 0.05). The levels of PGE2 produced by cells in fraction 1 were not statistically greater than those produced by fractions 4 + 5 when corrections were made for cell viability.
Whether or not the significant variations in PGE2 levels found among M@ fractions after 48 h culture are the result of differences in rates of synthesis or degradation is not directly demonstrated by these studies. However, after 30 minute incubations of the different M&?J fractions with [14Cl A.A. (figure l), different peak heights for PGE2 (as well as TXB2) are apparent. Furthermore, incubating the fractionated cells with 13~l-p~~2 for 30 minutes (the same incubation time used for the [14C] A.A. conversion studies) failed to show any detectable breakdown of the PGE2 (data not shown). These observations would suggest that differing activities of PG endoperoxide synthase and/or PG endoperoxide E isomerase, rather than differential catabolism of PGE2, account for the variations in PGE2 synthesis among the M8.
The differences in PGE2 synthesis among the M8 subpopulations could also be postulated to result from the differential activation of the cells by the fractionation procedure itself. By measuring the specific activities of the enzymes (5'-nucleotidaseand acid phosphatase) claimed to represent biochemical correlates of monocyte activation (14-16), fraction 1 cells were found to be more activated (i.e. lower 5'-nucleotidase levels, higher acid phosphatase levels) than cells sedimenting in fractions 3 or 4 + 5 (17). Therefore, no simple correlation was observed between the levels of PGE2 among the fractionated M# and their degree of activation as determined by enzyme analyses. Furthermore, when fraction 1 cells were incubated in concentrations of BSA comparable to those present in high density fractions (25%) and vice versa for 45 minutes (4'C) before carrying out the 48 h
cultures, there was no alteration
in their relative synthesis of PGE2.
These results appear to exclude
density gradient-related activation of the subpopulations as an explanation for the differential synthesis of PGE2.
The relatively low levels of PGE2 generated by fraction 4 + 5 could conceivably be attri"qltedto the lower viability and % M@ in this
692
NOVEMBER
1979 VOL. 18 NO. 5
PROSTAGLANDINS
fraction when compared to the other fractions. However, correcting for viability and % ML? in fraction 4 + 5 still gave levels of PGE2 (3.34 + 1.48 ng/106 cells) statistically lower than those in fraction 2 or 3 (p < 0.05). Therefore, cell viability does not account for the different levels of PGE2
produced by the MQifractions.
In summary, the data presented in this report document significant differences among human peripheral blood M@ subpopulationswith regard to the synthesis of PGE2 and PGEl.
If PGE2 synthesis represents a means
through which M@ can exert an immunoregulatory influence, our data support the existance of heterogeneity among these cells in their capacity to effect such PGE2-mediated regulation. It could be postulated that pathologically induced alterations in the relative numbers of the individual M@ subpopulations might lead to serious alterations in PGE2mediated immunoregulation. In addition, the profiles of TKA2 synthesis (as measured by TKB2) may reveal further heterogeneity among the human peripheral blood M@ fractions in regard to arachidonic acid metabolism. These possibilities are currently being explored.
ACKNOWLEDGEMENTS
We wish to thank Ms. M. Newton, Ms. K. Chinn and Mr. G. Benthin for their technical assistance. Drs. U. Axen and J. Pike of the Upjohn Co. (Kalamazoo, Mich.) kindly supplied the deuterium-labeled and standard prostaglandins. M.E.G. is the recipient of a Clinical Investigator Award from the National Institute of Arthritis, Metabolism, and Digestive Diseases. J.D.S. is an Investigator of the Howard Hughes Medical Institute. This work was supported by USPHS Grants AI-14014 and AI14572.
NOVEMBER 1979VOL.18NO.S
693
PROSTAGLANDINS
REFERENCES
1.
Pelus, L. and Strausser, H. Prostaglandins and the immune response. Life Sciences 20:903-913, 1977.
2.
Goldyne, M.E. Prostaglandins and the modulation of immunologic responses. Int. J. Dermatol. 16:701-712, 1977.
3.
Kurland, J.I. and Bockman, R.J. Prostaglandin E production by human blood monocytes and mouse peritoneal macrophages. J. Exp. Med. 147:952-957, 1978.
4.
Stockman, G.D. and Mumford, D.M. The effect of prostaglandins on the in vitro blastogenic response of human peripheral blood lymphocytes. Exp. Hematol. 2~65-72, 1974.
5.
Lichtenstein, L.M., Gillespie, E., Bourne, HR. and Henney, C.S. The effects of a series of prostaglandins on in vitro models of the allergic response and cellular immunity. Prostaglandins 2:519528, 1972.
6.
Bray, M.A., Gordon, D. and Morley, J. Regulation of lymphokine secretion by prostaglandins. Agents Actions 6:171-175, 1976.
7.
Goodwin, J.S., Messner, R.P., Bankhurst, A.D., Peake, G.T., Saiki, J.H. and Williams, R.C. Prostaglandin producing suppressor cells in Hodgkin's disease. N. Engl. J. Med. 297:963-968, 1978.
8.
Raff, H.V., Cochrum, K. and Stobo, J.D. Macrophage-T cell interactions in the Con A induction of human suppressor T cells. J. Immunol. 121:2311-2315, 1978.
9.
Raff, H.V. and Stobo, J.D.
Unpublished results.
10. Axen, U., Green, K., Horlin, D. and Samuelsson, B. Mass spectrometric determination of picomole amounts of prostaglandin E and using synthetic deuterium-labeled carriers. Biochem. B zophys. & . comm . 45:519-529, 1971. 11. Goldyne, M.E. and Hammarstrom, S. Quantitative determination of prostaglandins El and Fla by multiple ion analysis. Anal. Biochem. 88:675-681, 1978. 12.
Schlenk, H., Gellerman, J.L. Esterification of fatty acids with diazomethane on a small scale. Anal. Chem. 32(11): 1412-1414, 1960.
13. Morley, J., Bray, M.A., Jones, R.W., Nugteren, D.H. and Van Drop, D.A. Prostaglandin and thromboxane production by human and guinea pig macrophages and leukocytes. Prostaglandins 17:729-736, 1979.
694
NOVEMBER
1979 VOL. 18 NO. 5
PROSTAGLANDINS
14. North, R.J. The concept of the activated macrophage. J. 121:806-809, 1978.
Immunol.
15. Karnovsky, M.I. and Lazdins, J.K. Biochemical criteria for activated macrophages. J. Immunol. 121:809-813, 1978. 16. Cohn, Z.J. The activation of mononuclear phagocytes: Fact, fancy and future. J. Immunol. 121:813-816, 1978. 17. Picker, L., Raff, H.V., Goldyne, M.E. and Stobo, J.D. Biochemical heterogeneity among human monocytes. Submitted for publication. (Received 4/6/1979 Accepted 10/15/1079) Editor: Shozo Yamamoto
NOVEMBER
1979 VOL. 18 NO. 5
695