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

All

Copyright 0 1992 rights of reproduction

X/9?

54.00

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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