PROSTAGLANDINS

ANTIGEN-ANTIBODY COMPLEXES STIMULATE THE

SYNTHESIS AND RELEASE OF PROSTACLANDINS BY MOUSE PERITONEAL
MACROPHAGES

Robert J. Bonney, Peter Naruns, Philip Davies

and John L. Humes

Departments of Immunology
and Biochemistry of Inflammation

Merck Institute for Therapeutic Research

P.O. Box 2000
Rahway, New Jersey 07065

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PROSTAGLANDINS

SUMMARY

Antigen-antibody complexes (Ag/Ab) formed at equivalence stimulate the

release of arachidonic acid and synthesis of prostaglandin E2 and 6-keto-
prostaglandin F by resident mouse peritoneal macrophages. Prostaglandin
svnthesis and se cpetion is stimulated by submicrogram quantities of Ag/Ab which
increases in a dose-dependent manner. _This release is time-dependent -and occurs
in the absence of any loss of cell viability as indicated by increased cellular
levels of lactate dehydrogenase without concomitant loss of this activity to the
media and the continued secretion of a constitutive cellular product, lysozyme.
The stimulated synthesis of prostaglandins by Ag/Ab is inhibited by indomethacin
and physiological levels of antiinflammatory glucocorticoids.

INTRODUCTION

Macrophages respond to the products of stimulated T- and B-lymphocytes by

producing a number of inflammatory mediators. T-lymphocytes stimulated by
specific antigens or mitogens release macromolecules, named lymphokines or
lymphocyte activation products, which influence a number of macrophage
functions (for reviews, see 1, 2). These include migration inhibitory factor (31,
macrophage chemotactic factor (41, macrophage activating factor (51, and factors
which causes the selective release of lysosomal acid hydrolases (61, neutral
proteinases (7) and prostaglandins (8). The immunoglobulin products of 5
lymphocytes combine with specific antigens to form antigen-antibody complexes
that bind membrane Fc receptors on macrophages (9) and are subsequently
endocytosed (IO). Intradermal injection of antigen-antibody complexes formed
at equivalence into rats results in chronic inflammatory lesions (II). These
lesions are characterized by the presence of many macrophages which have
ingested the antigen-antibody complexes. Antigen-antibody complexes have also
been shown to stimulate the selective release of lysosomal hydrolases from
polymorphonuclear leukocytes (12) and macrophages (13). Recently we have shown
that mouse peritoneal macrophages exposed to the inflammatory particle,
zymosan, synthesize and release large amounts of prostaglandins and also release
lvsosomal acid hvdrolases in a selective manner (14). We now show that antigen-
antibody complexes stimulate the synthesis and release of prostaglandin E2 (PcE2)
and 6-keto-prostaglandin F (6KF ), the stable metabolite of PG12, by resident
mouse peritoneal macroph auges m &li tained in tissue culture.

MATERIALS AND METHODS

Tissue culture. Resident macrophages were collected by peritoneal lavage

from Male Swiss Webster (HLA-SW/ICR SPF Hilltop Lab Animals, Scottdale, Pa.)
The lavage medium used was Ml99 containing 100 units of penicillin and strepto-
mycin/ml, 20 units of heparin/ml, and 1% heat-inactivtted pig ser#m (56’C, 30
min). The cells were plated at a density of 4 x 10 - 5 x 10 per 50 mm
culture dish (Nunclon. Vanrrard Int.. Neotune. NJ.) and incubated for 2h at 370c
in 5% CO in air. ‘The lonadheient ‘cells’ were removed by washing the cell
sheet with2 five 5 ml volumes of phosphate buffered saline (PBS) using a
mechanized cell washing apparatus designed by P. D. Wightman of this laboratory
and the adherent cells maintained overnight in 4 ml Ml99 + 10% heat-inactivated

606 OCTOBER 1979 VOL. 18 NO. 4

 PROSTAGLANDINS

pig serum. Greater than 88% of the cells cultured in this way are identified
as macrophages. After 24h the medium was removed and the cells were washed
twice with 5 ml of PBS. Four ml of fresh Ml99 medium containine 1%
heat-inactivated pig serum and 1 uCi arachidonic acid ~,6,8,9,11,12,14,15-‘a, -61
Ci/mmole. from New England Nuclear, Boston. Mass. was added. The cells
were incubated for 4hr, &shed to remove the.remaining /‘H/-arachidonic acid
and fresh medium devoid of serum but ntaining the various stimuli was added.
Under these conditions the remaining P H&‘arachidonic acid is found exclusively
within cellular phospholipid and triglyceride (14). Antigen-antibody complexes
were prepared by adding equivalent amounts of rabbit anti-human serum albumin
(Cappel Laboratories, Inc., Cochranville, Pa.) to human serum albumin and
incubating the mixture at 37oC for Ih or room temperature overnight. The
complexes (Ag/Ab) were centrifuged, washed with PBS 3 times, and finally
suspended in culture medium. The concentration of Ag/Ab added to the cells
is expressed on the basis of the amount of antigen present. Zymosan was
purchased from K&K Labs Inc., Plainview, N.Y. and prepared as described (14).

Assay for 3H-arachidonate release.

Resident mouse peritoneal macroyhages were isolated by lavage as described

above and 0.2 ml containing 2 x 10 cells and 0.10 pCi of fl$arachidonic acid
was added to each well of a 96-well NUNC microtiter plate and incubated for
4 hr. The cells were then washed in PBS and fresh medium without serum but
containing 0.1% BSA, indomethacin (3pM) and either zymosan or Ag/Ab was
added. After 2 hr of incubation the radioactivity released into the medium
was determined. Under these conditions greater than 95% of the radioactivity
released into the media was co-chromatographic with authentic arachidonic acid
in a system composed of n-hexane/ethyl ether/acetic acid (60/40/l).

Harvest of media and cells for prostaglandin and lysosomal hydrolase assays.

At the termination of the experiments, media were collected and divided for
subsequent analysis. Two ml of 0.9% NaCl containing 0.1% Triton X-100 was
added to the plates and the lysed cells removed with a rubber “policeman”.

Enzyme assays.

Lactate dehydrogenase (LDH) was assayed by determining the rate of oxidation

of NADH at 340 nm. E-Acetyl-B-D-glucosaminidase (NAG) was assayed by the
method of Woolen et al. (15) with pnitrophenyl-N-acetyl-B-D-glucosaminide as
substrate dissolved z mM citric acid - sodium-phosphate, dibasic buffer, pH
4.5. A unit of enzyme activity for NAG is defined as the amount of enzyme
needed to cleave 1 nmol of substrate/h at 37OC. Lysozyme was assayed as
described by Gordon et al (16).

Extraction and chromatography of ‘H-labeled prostaglandins.

The harvested culture media were acidified with citric acid to 0.03M and
prostaglandins and arachidonic acid (30 ~1 were added as chromatographic
standards. The acidified media were then extracted as previously described (14).
The extracted materials were dissolved in 25 ul of ethyl acetate/methanol (3:1,
v/v), and quantitatively applied to 2 x 20 cm strips of SG-81 paper. The strips

OCTOBER 1979 VOL. 18 NO. 4 607

PROSTAGLANDINS

were developed by descending chromatography in the following system:

ethylacetate/acetic acid (99:1, v/v). The prostaglandin and fatty acid standards
were detected by iodine vapor. Quantitation was achieved by cutting the
chromatogram into small segments corresponding to the radioactivity peaks and
the radioactivity determined by counting the samples in 10 ml of Aquasol in a
Packard 3255 liquid scintillation spectrometer.

RESULTS

Figures 1 and 2 show that Ag/Ab prepared at equivalence stimulate the release
of arachidonic acid and the synthesis and secretion of prostaglandins from resident
mouse peritoneal macrophages maintained in culture.
.zymoCnon.
&ml

Fig. 1 Ag/Ab and zymosan stimulate the release of &7-arachidonic acid from
macrophage phospholipids. Resident macrophages were isolated and cultured in
microtiter plates as described in Materials and Methods. The amount of
radioactivity released into the media 2 hr after the addition of the stimulator
was determined The results are the mean f S.E.M., (N=6).

608 OCTOBER 1979 VOL. 18 NO. 4

 PROSTAGLANDINS

The proximal effect of the Ag/Ab is the stimulation of arachidq$c acid release
from membrane phospholipid. We have previously shown that L t-i_T-arachidonic
acid is incorporated into phosphatidylcholine, phosphatidylethanolamine and
triglycerides (53%, 31% and 16% respectively) in r sident mouse peritoneal
macrophages (14). Zymosan stimulates the release of r’ H$arachidonic acid from
phosphatidylcholine (14). F’ ure 1 shows that Ag/Ab have similar effects to
zymosan on the release30f P HI-arachidonic acid from prelabelled macrophages.
The accumulation of L l-&arachidonic acid in the medium is measured in the
presence of indomethacin (3uM) to block its conversion into prostaglandins. The
accumulation is dose-dependent with respect to the concentration of both Ag/Ab
and for zymosan. (Fig. 1).

Ag/Ab also stimulate the synthesis and secretion of PHI-PCE and &-$-6KF
in a dose-dependent manner (Fig. 2A). Stimulation of prostaglgndin synthesis ‘P
seen with as little as 0.4&ml Ag/Ab increasing in a dose-dependent manner to
IO&ml Ag/Ab, but with no er increment at 50kgg/ml
cause 20-25 fold increase in HjT-PGE2 and a IO-12 fold
synthesis and secretion.

1711 30
6. SelectiveEnzymeRelease

WI
I!
-: !5
2
20 p
.E

15 f
Q
f
IO e
“4 $

; of-I
0 2 4I 6I aI IO
I I,, 50 0 2I 4I 6Ii a IO
I I_&
0

Ag/Ab, pg/ml Ag/Ab, fig/ml

Fii 2.Resident macrophages were isolated and cultured as described in Materials

and Methods. Ag/Ab were added to 2 ml of serum-free Ml99 medium. Media
and cells were harvested after 4h of incubation and assayed for prostaglandin
and NAG content. The radioactivity cochromatographic with authentic PGE2
and 6KF,, was determined. The enzyme activity found in the culture media
divided by the total activity (cells plus media) is calculated as the percent of
activity found in the media. The results are the mean f S.D., N=3.

OCTOBER 1979 VOL. 18 NO. 4 609

PROSTAGLANDINS

Ag/Ab have been shown previously to stimulate the selective release of

lysosomal acid hydrolases by macrophages (13). Figure 2 shows that similar
concentrations of Ag/Ab stimulate the selective release of the lysosomal acid
hydrolase NAG and prostaglandins with maximal enzyme release also occurring
with lOpg/ml Ag/Ab. That the release of both prostaglandins and NAG is
occurring from viable cells is shown by measurement of the cytoplasmic enzyme
lactate dehydrogenase. Figure 28 shows that activity of this enzyme in the
culture medium does not increase with any of the concentrations of Ag/Ab used.
Also the cellular levels of this enzyme do not decrease, indeed they increase
considerably as shown previously (13) and Table 1. Another indicator of
macrophage viability under these experimental conditions is the unaltered rate
of secretion of lysozyme, a constitutive secretory product of mononuclear
phagocytes (16) and Table 1. This finding also indicates that Ag/Ab do not act
as a general stimulus of macrophage secretory activity.

TABLE 1

The Effect of Ag/Ab on Lysozyme Secretion and LDH Levels in

Cultures of Resident Macrophages

Additiona to Culture Lvsoznne Lactate Dehydrozenare

w nedia Cell6 Media

fug/culture) -W/ml)--

None 1.7 + 0.2 5.9 + 0.4 165 + 41 0

AglAb, 5 ug/m.l 2.4 2 0.3 5.12 0.7 145 + 52 0

AgIM, 10 ug/ml 2.4 + 0.0 4.9 + 0.6 324 + 28 0

AgfM, 25 rg/ml 2.9 f. 0.5 5.3 + 0.4 346 + 43 0

Macrophages were cultured for 24 hr as described in Materials and Methods.

Ag/Ab was added and the cells incubated for an additional 24 hr in serum free
media. The activity of lysozyme and LDH in the media and cell homogenates
were determined. * results are the mean + S.D. (N=3).

610 OCTOBER 1979 VOL. 18 NO. 4

 PROSTAGLANDINS

The synthesis and release of p@ -PGE F and ,&--6KF stimulated by Ag/Ab

occurs in a time-dependent manner ove 8 to 12 hours Big. 3). Figure 3 also
compares the effects of immune complexes and zymosan in this respect, showing
that the latter gives a greater maximal release of these products.

*
Lc
I I I 1 I
0 4 6 12 16 20 24

lime. hr.

Fig. 3. Resident macrophages were isolated and plated as described in Materials

and Methods. Twenty pg of Ag/Ab or IOOug of zymosan was added to 2 ml of
serum-free medium. Media and cells were harvested after various times of
incubation and assayed for prostaglandin content. The results are the mean f
S.D., (N=3).

OCTOBER 1979 VOL. 18 NO. 4 611

PROSTAGLANDINS

Antigen or antibody added alone to the macrophages did not stimulate the
release of lysosomal acid hydrolases and prostaglandins (Table 2). The amount
of Ag and Ab added to the dishes were the approximate amounts used to prepare
the complexes at equivalence.

TABLE 2

Ag/Ab but not Antigen or Antibody Alone Stimulate Prostaglandin

and NAG Release by Resident Macrophages

Additions Mediator Released

NAG
PCE2 6KFlcI (% in Media)
(CPM)

None 6542100 319*57 13k3

Ag/Ab, IOug/ml 2880_+153 1638+160 2827

Ag, IO&n1 316227 188+30 8+0.3

Ab, 930ug/ml 6532233 297~81 8+0.1

Resident macrophages were isolated from the peritoneal cavity of unstimulated mice
and cultured for 24h as described in Materials and Methods. Ag/Ab, antigen or antibody
alone were added and the cells incubated in serum-free Ml99 for 4h. Media and cells
were harvested and assayed for PC and NAG content. The radioactivity
cochromatographic with authentic PGE and 6KF was counted. The enzyme activity
‘found in the culture media divided by tzle total a & ivity (cells plus media) is calculated
as the percent of activity found in the media. The results are the mean 2 S.D. (N=3).

Both steroidal and eroidal antiinflammatory drugs inhibit the synthesis and
secretion of Y? I-&PGE and HJ-6KF by resident macpphages stimulated by Ag/Ab
(Table 3). Indomethaein at a concekation of 1 x 18 M inhibited the synthesis of
PCE and 6KF by 100% and 98% respectively. Three glucocortkoid antiinflammatory
dWa namely Bxamethasone, prednisolone and hydrocortisone inhibit FGE2 and 6KF
synthesis from 46% to 70%. The experiments were conducted with concentrations bn
the glucocorticoids found to give maximal inhibition. The effect of glucocorticoids
were found to be both dose-dependent and also to require a 1 hour preincubation period
for expression of maximal activity. The inhibition of prostaglandin synthesis and
secretion by the steroids was not due to toxic affects on the cells since they did not
cause an increased release of LDH into the media.

612 OCTOBER 1979 VOL. 18 NO. 4

 PROSTAGLANDINS

TABLE 3

Effect of Antiinflammatory Drugs on Prostaglandin Production

Stimulated by Ag/Ab

Additions Cont. PG Released

VJJ- (CPM)

PGE2 %Inhib. %Inhib.

6KFlo
None 707kl41 275253 3.19.9

Ag/Ab, iOug/ml - 17,496+451 2734k277 2.822.7

-8
Ag’Ab+Dexamethasone 10 9721+408 (46%) 1365+_112 (56%) 1.3t2.6

Ag/Ab+Dexamethasone IO6 6186+211 (67%) 1013+_98 (70%) 5.4t3.6

-6
Ag/Ab+Pre&isolone 10 6921i1465 (63%) iO21+_78 (70%) 4.2t3.2
-6
Ag/Ab+Hydrocortisone 10 10276 tiO76 (43%) 1541*57 (48%) 2.8t4.1
-6 73Q64 317+35
Ag/Ab+Indomethacin 10 (100%) (98%) 2.4tO.6

Macrophages were cultured as described in Materials and Methods. The drugs

were dissolved in DMSO and added to the culture media. After 1 h of incubation,
IOugg/ml of Ag/Ab were added and the cells incubated for an additional 4 h.
Media and cells were harvested and assayed for prostaglandin and LDH content.
The results are the mean ?r S.D. (N=3).

DISCUSSION

The formation and deposition of Ag/Ab at sites of acute and chronic

inflammation is thought to play a maj& role in the pathogenesis of various
diseases. This is mediated in part by the interaction of Ag/Ab with phagocytic
cells, such as polymorphonuclear leukocytes and macrophages, by specific
interaction with Fc receptors found on the surface of these cells. The binding
of antigen-antibody complexes to these Fc receptors stimulates phagocytosis.
In some instances the phagocytic process is accompanied by the release of
inflammatory mediators such as lysosomal acid hydrolases (12-13).

macrophages responding to inflammatory stimuli such as zymosan (14) or phorbol

myristate acetate (17). Similar findings for PGE production by macrophage
exposed to antibody-coated erythrocytes have been made by Glatt et al (18).
The present studies document that arachidonic acid oxygenation products are
synthesized and released by macrophages responding to Ag/Ab. Antibody or
antigen alone does not stimulate the synthesis and secretion of arachidonate
oxygenation products. The initial effect 8f the Ag/Ab as well as zymosan
appears to be the increased release of L HJ arachidonic acid from cellular

OCTOBER 1979 VOL. 18 NO. 4 613

PROSTAGLANDINS

phospholipid. As is seen in Figure 1 increased amounts of ,&$arachidonic acid

can be trapped by bovine serum albumin included in the culture medium of
macrophages exposed to Ag/Ab OT zymosan. Such release is stimulated in a
dose-dependent manner by similar concentrations of these stimuli to those which
trigger the synthesis of arachidonic acid oxygenation products (Figure 2 and 3).
Once released the arachidonic acid is converted into two primary products, F’GE
and 6KF . This stimulated secretion of prostaglandin due to the Ag/Ab i?J
inhibited’8y both nonsteroidal and steroidal antiinflammatory agents.

In view of the observations of Gordon et al (8) that lymphokines stimulate

the synthesis of prostaglandins by macrophages it appears that the products of
both T and B lymphocytes stimulate the synthesis of prostaglandins by this cell
type. The significance of such a stimulation in relation to the overall importance
of lymphocyte-macrophage interactions as part of the effector arm of host
defence responses and inflammatory reactions remains to be determined.

614 OCTOBER 1979 VOL. 18 NO. 4

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REFERENCES

1. Allison, A. C., C. Cardella and P. Davies, 1976. Immune complexes and

induced release of lysosomal enzymes from mononuclear phagocytes in the
pathogenesis of rheumatoid arthritis. In: Immunological Aspects of
Rheumatoid Arthritis, Pharmacology, Vol. 6TKarger, Basel, p. 251.

2. Cohen, S. 1977. The role of cell-mediated immunity in the induction of

inflammatory responses. -In: The Am. J. Path. -88: 502.
3. David, J. and David, R. A. 1972. Cell hypersensitivity and immunity. Prog.
Allergy -16: 300.

4. Ward, P. A., H. G. Remold and J. R. David, 1970. The production by

antigen-stimulated lymphocytes of a leukotactic factor distinct from
migration inhibitory factor. Cell. Immunol. & 162.

5. Nathan, C. F., M. L. Karnovsky and J. R. David, 1971. Alterations of

macrophage functions by mediators from lymphocytes. J. Exp. Med. 133:
1356.

6. Pantalone, R. M. and R. C. Page, 1975. Lymphokine-induced production and

release of lysosomal enzymes by macrophages. Proc. Nat. Acad. Sci. U.S.A.
-72: 2071.
7. Gordon, S. and Cohn, 2. A., 1978. Bacille Calmette-Guerin infection in the
mouse. Regulation of macrophage plasminogen activator by T-lymphocytes
and specific antigen. J. Exp. Med. -147: 1175.

8. Gordon, D., M. A. Bray and J. Morley, 1976. Control of lymphokine production

by prostaglandins. Nature -262: 401.

9. Hay, F. C., G. Torrigiani and I. M. Roitt, 1972. The binding of human IgG
subclasses to human monocytes. Eruop. J. Immunol. 2: 257.

10. Steinman, R. M. and Z. A. Cohn, 1972. The interaction of particulate

horseradish peroxidase (HRP) anti-HRP immune complexes with mouse
peritoneal complexes in -- vitro. J. Cell Biol. II: 616.

11. Spector, W. G. and N. Heesom, 1969. The production of granulomata by

antigen-antibody complexes. J. Path. 98: 31.

12. Henson, P. M. 1971. Interaction of cells with immune complexes, adherence,

release of constituents and tissue injury. J. Exp. Med. -134: 1145.

13. Cardella, C. J.. P. Davies and A. C. Allison, 1974. Immune complexes

induce s&ctive’release of lysosomal hydrolases’from macrophages --
in iitro..
Nature -247: 46.

14. Bonney, R. J., P. D. Wightman, P. Davies, S. J. Sadowski, F. A. Kuehl Jr.

and J. L. Humes, 1978. Regulation of prostaglandin synthesis and of the
selective release of lysosomal hydrolases by mouse peritoneal macrophages.
Biochemical J. 176: 433.

OCTOBER 1979 VOL. 18 NO. 4 615

PROSTAGLANDINS

15. Woolen, J. W., R. Heyworth and D. G. Walker, 1960. Studies on

glucosaminidase and N-acetyl- BOD-glucosaminidase. Biochem. J. -78: 111.

16. Gordon, S., J. 0. Todd and 2. A. Cohn, 1974. --

In vitro synthesis and secretion
of lysozyme by mononuclear phagocytes. J. Exp. Med. -13% 1228.

17. Humes, J. L., P. Davies, R. J. Bonney and F. A. Kuehl, Jr., 1978. Phorbol
myristate acetate stimulates the release of arachidonic acid and it cyclo-
oxygenase products by macrophages. Fed. Proc. x: 1318.

18. Glatt, M., H. Kafin, K. Wagner and K. Brune, 1977. Prostaglandin release
from macrophages: An assay system for anti-inflammatory drugs --
in vitro.
Agents and Actions 7; 321.

616 OCTOBER 1979 VOL. 18 NO. 4