Lipopolysaccharide induces prostaglandin H synthase-2 in alveolar macrophages
Descripción
Vol. 187, No. 2, 1992 September
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
16, 1992
Pages
Lipopolysaccharide
Induces
Prostaglandin
in Alveolar
M.G.
O’Sullivan’*,
E.M.Huggins
‘Department
Jr.‘,
of Medicine,
E.A. Meade2,
Wake
Forest
August
H Synthase-2
D.L Dewitt2
University 042
Michigan
State
of Biochemistry,
and C.E. McCall’
Medical
NC 27 157-I
East Lansing, Received
127
Macrophages
Winston-Salem, 2Department
1123-l
Center
University
Ml
7, 1992
Prostaglandin H synthase is a key enzyme in the formation of prostaglandins and thromboxane from arachidonic acid. The recent cloning of a second prostaglandin H which is distinct from the classic synthase gene, prostaglandin H synthase-2, prostaglandin H synthase-1 gene, may dramatically alter our concept of how cells regulate prostanoid formation. We have recently shown that the enhanced production of prostanoids by lipopolysaccharide-primed alveolar macrophages involves the induction of a novel prostaglandin H synthase (J. Biol. Chem., (19921, 267, 14547-14550). We report here that the novel PGH synthase induced by lipopolysaccharide in alveolar ID1992 AcadernlcPress, Inc. macrophages is prostaglandin H synthase-2.
The
stimulus-induced
coordinated synthase)
activity (reviewed
membrane
in turn,
by Smith after
acid
is then
is metabolized
thromboxane
synthase)
thromboxane,
respectively).
prostanoid acid
cascade.
prostanoid to hereafter
*To
synthesis
whom
An
formation
of prostanoids
of phospholipase
phospholipids
Free arachidonic which,
formation
eta/.
A,
ligand-receptor metabolized
yield
enzymes
as PGH synthase-2)
(3-5)
should
Abbreviations. LPS: lipopolysaccharide;
activation
is a potentially
is distinct
and
H synthase
(PGH
acid is cleaved
from
to form
H,,
E,-isomerase
and
important
how
A,.
prostaglandin
prostaglandin
E,
and
determinant
at an early point
of a second
sequential
of phospholipase
(e.g.
in understanding
which
(PG)
(e.g. prostaglandin
of its position
cloning
the
Arachidonic
prostanoids
development
was the recent
(2)).
by PGH synthase
PGH synthase
exciting
involves
prostaglandin
coupled
specific
by a cell because
correspondence
and
(I 1, and Dewitt
by specific to
by cells
of
in the arachidonic cells
PGH synthase
from the classic
may
regulate
gene
(referred
PGH synthase-1
be addressed. PGH synthase:
prostaglandin
H synthase. 0006-291
1123
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Copyright 0 1992 rights of reproduction
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by Academic Press. Inc. in ally form reserved.
Vol.
187,
cloned
No.
BIOCHEMICAL
2, 1992
from sheep,
cells by diverse
mouse
processes
AND
and human including
BIOPHYSICAL
cells (1,6).
mitogenesis,
RESEARCH
PGH synthase-2 transformation
COMMUNICATIONS
is rapidly
and serum
induced
treatment
in (3-
5). Lipopolysaccharide and especially macrophages
enhances (7,8).
enhanced
formation
opsonized
zymosan
induction
of a novel
the
novel
(LPS) increases
PGH
their
formation
We have
shown
of prostanoids (9,lO).
synthase
induced
of prostanoids
by inflammatory that
LPS primes
in response
Moreover,
PGH synthase
the formation
this
by LPS
alveolar
to the
increased
in LPS-primed
cells such
is PGH
as monocytes
macrophages
receptor-mediated
prostanoid
AM (9).
by cells in general,
synthase-2
for
stimulus
synthesis
In this report,
(AM)
and
involves
the
we show
that
by Western
blot
and
immunoprecipitation. Materials
and Methods
Reagents Zymosan, protease inhibitors, and protein-A-coupled sepharose beads were from Sigma Chemical Co. (St. Louis, MO). Fetal calf serum was from HyClone (Logan, UT), Ml 99 and RPM1 media were from Whittaker, M.A. Bioproducts (Walkersville, MD). LPS, derived from Escherichia co/i 0111 :B4, was from Difco Laboratories, Detroit, Ml. lsozyme specific antisera for PGH synthase-2 was produced by immunizing rabbits with the synthetic 17-mer peptide; cys-tyr-ser-his-ser-arg-leu-asp-asp-iso-asn-pro-thr-val-leu-is~ lys (DD21, coupled to keyhole limpet hemocyanin. This peptide is derived from a unique region of PGH synthase-2 protein near the carboxy-terminal which is not present in PGH synthase-1. The amino-terminal cysteine of this peptide was added to allow coupling to maleimide-activated keyhole limpet hemocyanin, and is not derived from the PGH synthase-2 sequence. Anti-PGH synthase-1 antibody was obtained from Cayman Chemical Co., Ann Arbor, Ml. Microsomes from cos-1 cells transfected with PGHsynthase-2 were prepared as described (1 I). Collection and preparation of alveolar macrophages Bronchoalveolar lavage was performed on lungs humanely removed from 3 kg New Zealand White rabbits, as previously described (9, IO). Alveolar macrophages were suspended in Ml 99 or RPM1 medium containing 10% fetal bovine serum and antibiotics (9), and plated in 6-well plates or in 75 cm2 flasks. After addition of LPS (1 rig/ml), the cells were cultured for 4 h at 37OC in 5% CO,, 95% air. The alveolar macrophages adhered to the flask surface during this period. Westernblot analysis Alveolar macrophages were cultured with LPS in 75 cm2 flasks for 4 h, and lysed with ice-cold lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 10 mM EDTA, 1% NP40, 0.1% SDS, 1 mM PMSF, aprotinin, 1 pug/ml, pepstatin 1 pg/ml). After centrifugation (12,OOOxg / 15 min), samples of supernatant (adjusted for equal amounts of protein) were separated by sodium dodecyl sulfate-lo% polyacrylamide gel electrophoresis. Proteins were electroblotted onto Hybond-ECL nitrocellulose membranes (Amersham Corp., Arlington Heights, IL) , and treated with blocking buffer before 1 h incubation at 20°C with anti-DD2 antibody. Chemiluminescence of luminol oxidized by horseradish peroxidase-conjugated goat anti-rabbit IgG was detected by Hyperfilm-ECL film (Amersham Corp., Arlington Heights, IL). t’mmunoprecipitation of /35SJ-labeled prostagfandin H synthase Alveolar macrophages were cultured with LPS for 4 h in 6-well plates (3x1 06/weII) in methionine-free RPMI-1640 containing 135Sl-methionine (100 FCilwell) (Tran 35S-Label, 1 147 Ci/mmoL ICN Radiochemicals, Irvine, CA) and 10% fetal bovine serum. Macrophages were washed, and lysed in lysis buffer as above. After centrifugation (12,OOOxg / 15 min) and liquid scintillation spectrometry, samples of supernatant were adjusted to equal cpm, and 1124
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
immunoprecipitation carried out using anti-DD2 antibody or anti-PGH synthase-1 antibody at 4OC overnight. Cold competition experiments were conducted by addition of 100 pg of the 17 amino acid synthetic peptide DD2 (unique to PGH synthase-21, 170 ,ug of PGH synthase-2 microsomes from cos-1-transfected cells, or 5 pg of authentic sheep PGH synthase-1 (Oxford Biomedical Co., Oxford, MI). Bound antigen-antibody complexes were recovered using protein-A-coupled sepharose beads and immunoprecipitated proteins were separated by sodium dodecyl sulfate-l 0% polyacrylamide gel elecrophoresis. After electrophoresis, gels were fixed, dried and prepared for fluorography at -7OOC using Kodak XAR-5 film. Results
and Discussion
Alveolar
macrophages
approximately
that
72 kDa protein
an antibody
(anti-DD2)
synthase-2
were
raised
doublet against
but not in PGH synthase-1
derived
PGH synthase-2
alveolar
macrophage
PGH synthase-2,
(Fig 1).
macrophages
is PGH synthase-2
peptide
and of microsomes
DD2,
Additional
out immunoprecipitation
with
by western a unique
blot and amino
different possibly
evidence
that
is indicated from
LPS showed
migration indicating
PGHS-1 “: :
01
123
4
-.
M
B P
in alveolar synthetic
PGH synthase-2,
LID-2
‘i ln
COMPETE 7 “: F
H
:
::
7 : x
‘80 .49.5
Figure 1. Western blot of LPS-primed alveolar macrophage proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using anti-DD2 antiserum. There is marked induction of a protein doublet (- 72 kDa ) in LPS-treated (1 rig/ml for 4 h) alveolar macrophages (lane 4) compared to control macrophages (lane 3). Lane 1: 10 n9 PGH synthase-1, Lane 2: 5 /JQ microsomes from PGH synthase-2 transfected cos-1 cells. Positions of 49.5- and 80-kDa molecular mass marker proteins are indicated in the margin. Figure 2. Autoradiograph of immunoprecipitated, [35S1-labeled, alveolar macrophage proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lanes all other lanes represent LPS-treated 1 and 7 represent control macrophages; macrophages. Lanes l-5 and 7-l 1 were immunoprecipitated with anti-PGH synthase-1 antibody and anti-DD2 antibody, respectively. Lanes 3-5 and 9-l 1 represent competition attempts with DD2 synthetic peptide (100 /lg) (lanes 3 and 91, microsomes (17Op9) from PGH synthase-2 transfected cos-1 cells (lanes 4 and lo), and authentic PGH synthase-1 (5 fig) (lanes 5 and 11). There is marked induction of a protein doublet at approximately 72 kDa in LPS-treated (lng/ml for 4 h) cells (lanes 2 and 8) compared to control alveolar macrophages (lanes 1 and 7). Lane 6 represents 100, 97, 69 and 46-kDa molecular mass marker proteins, whose positions are indicated in the margin. 1125
to
(Fig 2, lanes 9 and IO).
ANTI
COMPETE
cos-l-cell
post-translational
of the 17 amino-acid with
in PGH
LPS-induced
PGH synthase
PGH synthase
P
using
that
than
different
LPS-induced
A N1I
of an
present
noting
pattern
cells transfected
of LPS-induced
induction
immunoprecipitation,
It is worth
by the ability
cos-1
marked
acid sequence
(Figs 1 and 2).
has a slightly
glycosylation
compete
incubated
Vol.
187,
No.
In contrast,
BIOCHEMICAL
2, 1992
authentic
PGH synthase-I
induced
PGH synthase
reported
that LPS rapidly
probe
for the
synthase induced
mRNA
reports
using
murine
enzymatic
induces PGH
mass (9).
evidence
alveolar
macrophages.
A second
finding
in our present
antibody recognized
doublet
against
have shown
transfected
cos-1
to compete
anti-PGH
PGH synthase-2 rigorous
immunological
transfected reflect
may share
cos-1
addition
cells to compete
of insufficient
of anti-PGH
synthase-1
PGH synthase-2.
Endotoxemia
is characterized and thromboxane,
findings,
on this study
is responsible
for
macrophages,
suggests
leukocyte
prostanoid
PGH synthase-2 of prostanoids induction
the
that
formation.
in leukocytes observed
cytokine,
Furthermore, and other
PGH
because
PGH
a more
synthase-2
of possible
from
lower
affinity
synthase-2
than
of inflammation,
for
including
(13,14).
Our
of PGH synthase-2
by
LPS-primed
may
be an important
it raises
the possibility
cells may be responsible
alveolar
determinant that
of these
respective
has recently
been reported
in human
monocytes
of PGH synthase
of
induction
for the elevated
In support
1126
and
(Fig 2, lane 4) may
(91, that induction
(5), and induction
of DD2
PGH synthase-1
in endotoxemia.
interleukin-1
from
of this will require
of prostanoids
synthase-2
synthase-
PGH synthase-2
to its pathophysiology
studies
the protein
The failure
PGH synthase-2
cell-derived
contribute
to that
PGH synthase-2
of microsomal
protein,
synthesis
PGH
proof
levels of mediators
and previous
enhanced
of PGH synthase-2
the inflammatory
that
doublet
The anti-PGH
of LPS-induced
Definitive
cos-1
by elevated
prostaglandins based
for
a polyclonal
also recognize
blots (9,12).
out LPS-induced
antiserum
macrophage
antisera
(Fig 2, lane 3) suggests
PGH synthase-2
that
(Fig 2, lanes 2 and 8, and (9)).
observations).
failure
the present
PGH synthase-
protein
(unpublished
The
with
of PGH synthase-2
raised against
microsomal
epitopes.
analysis.
are consistent
homologue
also recognizes
antibody
PGH
the size of the
findings,
the identical
in western
enhances
Moreover,
on the observation
synthase-1
blots
common
to a cDNA
doublet
antisera
out immunoprecipitation
synthase-1
that hybridizes
our previous
the rabbit
recognizes
study
previously
(9).
in immunoprecipitations
cells rn western
peptide
with
is based
of PGH synthase-2
used in the present
using
This
that anti-PGH
1 antiserum
when
together
of LPS-
1 1). We have
LPS markedly
protein
study is that certain
antiserum
characteristic
synthetic
Taken
PGH synthase-1
by anti-DD2
We and others
of the induced
PGH synthase-2.
raised
that
macrophages
that LPS induces
in rabbit
1 also recognize
and
COMMUNICATIONS
out immunoprecipitation
4.3 kb mRNA
gene,
alveolar
RESEARCH
(Fig 2, lane
approximately
in rabbit
for PGH synthase-2 direct
antiserum
synthase-2
activity
BIOPHYSICAL
fails to compete
anti-DD2
and molecular
study provides
AND
of
levels
hypotheses, exposed
to
has been
Vol.
187,
No.
observed
BIOCHEMICAL
2, 1992
in macrophages
findings
from
an important
a number
isolated
from
of different
role in endotoxemia
AND
BIOPHYSICAL
endotoxemic
laboratories
suggest
and in a variety
RESEARCH
mice
(15).
COMMUNICATIONS
Collectively,
that PGH synthase-2
of inflammatory
these may play
conditions.
Acknowledgments This work
was supported
and by the American
by National
Institutes
of Health
Grants
HL29293
and AI-091
69,
Lung Association.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12. 13. 14. 15.
Smith, W.L., Marnett, L.J., and De Witt, D.L. (1991) Pharmac. Ther. 49, 153-l 79. Dewitt, D.L. (1991) Biochim. Biophys. Acta 1083, 121-134. Xie, W., Chipman, J.G., Robertson, D.L., Erikson, R.L., and Simmons, D.L. (1991) Proc. Natl. Acad. Sci. USA 88, 2692-2696. Kujubu, D.A., Fletcher, B.S., Varnum, B.C., Lim, R.W., and Herschman, H.R. (1991) J. Biol. Chem. 266, 12866-12872. O’Banion, M.K., Winn, V.D., and Young, D.A. (1992) Proc. Natl. Acad. Sci. USA 89, 4888-4892. Dewitt, D.L., El-Harith, E.A., Kraemer, S.A., Andrews, M.J., Yao, E.F., Armstrong, R.L., and Smith, W.L. (1990) J. Biol. Chem. 265, 5192-5198. Fu, J-Y, Masferrer, J.L., Seibert, K., Raz, A., and Needleman, P. (1990) J. Biol. Chem. 265, 16737-l 6740. Aderem, A.A., Cohen, D.S., Wright, S.D., and Cohn, Z.A (1986) J. Exp. Med. 164, 165-179. O’Sullivan, M.G., Chilton, F.H., Huggins, E.M. Jr., and McCall, C.E. (1992) J. Biol. Chem. 267,14547-14550. O’Sullivan, M.G., Fleisher, L.N., Olson, N.C., MacLachlan, N.J., and Brown, T.T. (1990) Am. J. Vet. Res. 51, 1820-1825. Meade, E.A., Smith, W.L., and Dewitt, D.L. (1992) In Prostaglandins, Thromboxanes, Leukotrienes and Related Compounds: Update 1992 (L.S. Wolfe and A.W. Ford-Hutchinson, Eds.), In Press, Elsevier Science Publishers, The Netherlands. O’Banion, M.K., Sadowski, H.B., Winn, V., and Young, D.A. (1991) J. Biol. Chem. 266, 2326 l-23267. Bone, R.C. (1991) Ann. Intern. Med. 115, 457-469. Olson, N.C., Salzer, W.L., and McCall, C.E. (1988) Mol. Aspects Med. 10, 511629. Masferrer, J.L., Seibert, K., Zweifel, B., and Needleman, P. (1992) Proc. Natl. Acad. Sci. USA 89, 3917-3921.
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