Full text of DOI:10.1007/PL00000224

©
Birkhäuser Verlag, Basel, 2001
Inflamm. res. 50 (2001) 496–499
023-3830/01/100496-04 $ 1.50+0.20/0
1
Inflammation Research
CD44-mediated cyclooxygenase-2 expression and thromboxane A2
production in RAW 264.7 macrophages
L. K. Sun*, P. Wahl*, G. Bilic and R.P. Wüthrich
Division of Nephrology, Department of Medicine, Kantonsspital, Rorschacherstrasse 95, CH-9007 St. Gallen, Switzerland, Fax: ++41 71 494 28 77,
e-mail: rpw@kssg.ch
Received 12 March 2001; returned for revision 18 April 2001; accepted by G. Geisslinger 12 June 2001
Abstract. Objective and design: CD44 is the major cell sur-
face receptor for hyaluronan (HA) on macrophages. Stimula-
tion of macrophages via the HA-CD44 pathway leads to the
enhanced expression of inflammatory gene products, includ-
ing cytokines, chemokines, and adhesion molecules. We have
examined whether activation of CD44 by crosslinking is
capable of activating the cyclooxygenase (COX) and prosta-
glandin (PG)/thromboxane (TX) pathway in cultured macro-
phages.
that these HA fragments display important proinflammatory
effects on macrophages [1]. The cell surface glycoprotein
CD44 is the principal receptor for HA on macrophages [2].
Activation of the CD44 pathway leads to the nuclear translo-
cation of NF-kB with subsequent transcription of cytokines
and other proinflammatory genes [3–5].
Prostaglandins (PGs) and thromboxanes (TXs) are
important autacoids, which modulate inflammatory reac-
tions. Cyclooxygenase-2 (COX-2) is the enzyme, which is
primarily responsible for the induced production of PGs and
TXs [6, 7]. Enhanced expression of COX-2 has been found at
inflammatory sites in animals and in patients with inflamma-
Materials and methods: CD44 was crosslinked on RAW
264.7 mouse macrophages using specific rat anti-mouse
CD44 monoclonal antibodies and anti-rat IgG. Total RNA
was extracted and subjected to RT-PCR analysis for genes of
the PG/TX synthetic pathway. Supernatants were analyzed
for PGE and TXB using specific ELISAs.
tory diseases [8, 9]. TXA
eicosanoids, which have been implicated in the regulation of
inflammatory and immunological events, TXA having a
proinflammatory and vasoconstrictory role, and PGE having
and PGE are two important
2
2
2
2
2
Results: Transcripts for COX-1, COX-2, TX synthase (TXS),
2
and PGE synthase (PGES) were all constitutively expressed
an anti-inflammatory and vasodilatatory function. In a previ-
ous report we found that stimulation of RAW 264.7 mouse
macrophages with HA fragments enhanced COX-2 mRNA
2
in the mouse macrophage cell line RAW 264.7. Crosslinking
of CD44 markedly enhanced COX-2 and weakly increased
TXS mRNA, whereas COX-1 and PGES mRNA did not
change significantly in these cells. Crosslinking of CD44
selectively increased the production of TXB but not PGE .
transcript levels and TXA production [10]. The purpose of
2
the present work was to elucidate the role of direct CD44
activation via receptor crosslinking on COX-2 expression
2
2
Conclusions: These findings suggest that the activation of
the CD44 pathway plays a unique role in PG synthesis.
and the subsequent differential production of TXA
2
and
PGE
.
2
Activation of this pathway results in enhanced TXA but
2
not PGE production. This leads to an imbalance of the
2
TXA /PGE profile which favors a proinflammatory and
Materials and methods
2
2
vasoconstrictory response.
Cell culture and stimulation protocol
Key words: Cyclooxygenase-2 – CD44 – Hyaluronan –
Macrophages
The mouse monocyte-macrophage cell line RAW 264.7 was grown in
DMEM supplemented with 10% FBS, 10 mM HEPES, 100 U/ml peni-
cillin and 100 mg/ml streptomycin. After resting the cells overnight in
DMEM/1% FBS, cells were incubated for 30 min with different rat anti-
mouse CD44 monoclonal antibodies (mAbs) at 10 mg/ml, including
Introduction
clones IRAWB14.4 (an activating IgG2 mAb [2]) and IM7.8.1 (an inert
a
IgG2b mAb). In control experiments cells were also incubated with anti-
CD49d (VLA-4a) mAb (clone R1-2 [IgG ]), anti-CD16/CD23 (Fcg
Upon degradation, high mol. wt. hyaluronan (HA) is frag-
mented into intermediate mol. wt. products. It has been shown
2b
receptor) mAb (clone 2.4G2 [IgG ]), or anti-CD90 (Thy-1.2) mAb
2b
(
clone 30-H12 [IgG ]) for 30 min. The IRAWB14.4. mAb was a gift
2b
from J. Lesley, the other mAbs were purchased from Pharmingen (San
Diego, CA). After washing away excess antibody, cells were then
exposed to anti-rat IgG (5 mg/ml) for different time intervals. Stimula-
Correspondence to: R.P. Wüthrich
*
Likang Sun and Patricia Wahl contributed equally to this work.

Vol. 50, 2001
CD44-mediated cyclooxygenase-2 expression and thromboxane A production in RAW 264.7 macrophages
497
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tion with intermediate mol. wt. HA (derived from human umbilical
cords; 100 mg/ml; Fluka, Buchs, Switzerland) and LPS (derived from E.
coli serotype 055:B5; 100 ng/ml; Sigma, St. Louis, MO) were used as
positive controls.
Results
Characterization of the RAW 264.7 cell surface
Fig. 1 demonstrates that RAW 264.7 cells express CD44
Flow cytometry
abundantly. RAW 264.7 also express Fcg receptors andVLA-
4
, but lack Thy-1.2. RAW 264.7 cells therefore display a cell
6
Cells (1¥10 ) were washed once with PBS containing 2% FBS, and
surface molecule profile which is typical of macrophages.
incubated with primary mAbs at 10 mg/ml for 1 h at 4°C. Cells were
washed and incubated with FITC-conjugated goat anti-rat IgG (Sigma,
St. Louis, MO) at 1:100 in PBS/2% FBS for 1 h at 4°C. After repeated
washing, cells were analyzed using a Becton Dickinson flow cytometer
Effect of CD44 crosslinking on PG synthesizing enzyme
expression
®
and the Cell Quest software (Becton Dickinson, San Jose, CA).
We then examined the effect of CD44 crosslinking
(IRAWB14.4 mAb) on COX-1, COX-2, TXS, and PGES
mRNA expression in RAW 264.7 cells by RT-PCR analysis.
Fig. 2A demonstrates that COX-1 and PGES mRNA levels
did not change significantly, whereas COX-2 mRNA was
significantly enhanced. TXS mRNA was also weakly
enhanced in response to CD44 crosslinking. In a control
experiment, crosslinking of another macrophage cell surface
RNA extraction and RT-PCR analysis for COX-1, COX-2,
TXS, and PGES mRNA
After crosslinking, total RNA was extracted from RAW 264.7 cells with
Trizol® reagent (Life Technologies, Gaithersburg, MD) and
phenol/chloroform extraction according to the manufacturer’s instruc-
tions. The RNA was quantified, lyophilized, and analyzed for COX-1,
COX-2, TXS and PGES expression by semi-quantitative RT-PCR, using
the Access RT-PCR kit from Promega (Madison, WI). The primer
sequences for mouse COX-1 (450 bp fragment), COX-2 (771 bp frag-
ment), TXS (360 bp fragment) were as described [10]. The primer
sequences for mouse PGES were as follows: forward primer 5¢-TTT
CTG CTC TGC AGC ACA CT-3¢, reverse primer 5¢-GAG GAA ATG
TAT CCA GGC GA-3¢ (267 bp fragment) [11,12]. The housekeeping
gene GAPDH was also amplified to control for equal amounts of
RNA (516 bp fragment), as described [13]. One mg total RNA was
reverse transcribed at 48°C for 45 min, followed by the PCR reaction.
The cycling parameters were as follows: denaturation at 94°C for
receptor, VLA-4 (a b integrin) enhanced COX-2 and PGES,
1
but not COX-1 and not TXS mRNA (Fig. 2B). Treatment
with LPS enhanced COX-2, TXS and PGES, but not COX-1.
Control stimulation by primary mAb and secondary antibody
alone were also performed, showing no effect on all four gene
transcript levels (data not shown).
Fig. 3 shows that COX-2 protein levels also increased in
RAW 264.7 cells after crosslinking with CD44. COX-2 pro-
tein expression was strongly enhanced after 1 h and 2 h,
decreasing again after 4 h. As shown before [10], this up-reg-
ulation was comparable with LPS and HA stimulation. Inter-
estingly, the IRAWB14.4 anti-CD44 mAb alone also stimu-
lated COX-2 protein. This could be a post-transcriptional
3
2
0 s, annealing at 50°C to 57 °C for 1 min, extension at 68°C for
min over 25–40 cycles, followed by the final extension at 68°C
for 7 min. Amplification was linear between 25 and 35 cycles, and at
RT dilutions between 1:10 and 1:1000. The RT-PCR was performed
with a Gene Amp PCR System 9700 thermocycler (Perkin-Elmer,
Foster City, CA). RT-PCR products were resolved on 1% agarose gels
and stained with ethidium bromide. Gels were then photographed with
UV light.
Protein extraction and western blotting for COX-2
After crosslinking, RAW 264.7 cells were washed twice with ice-cold
PBS and lysed in 1 ml of ice-cold extraction buffer (50 mM Tris-HCl,
pH 7.4, 1% NP-40, 1 mM EDTA, 1 mM PMSF and 1 ¥ protease
inhibitor cocktail [Sigma]). After 30 min, the cells were scraped off and
centrifuged at 10,000 rpm for 20 min at 4°C. The supernatants were
mixed with 4¥ non-reduced sample buffer (0.38 M Tris base, 8% SDS,
4
mM EDTA, 40% glycerol and 10 mg/ml bromophenol blue, pH 6.8),
and heated for 5 min. Equal amounts of protein extracts were separated
by using 10% SDS-PAGE. Proteins were then transferred to nitrocellu-
lose membranes, and COX-2 was detected with a chemiluminescent
detection system (Tropix, Bedford, MA), using the affinity-purified
polyclonal rabbit anti-murine COX-2 Ab (Cayman, Ann Arbor, MI).
TXB and PGE assays
2
2
After crosslinking cells overnight in DMEM/1% FBS, the content of
TXB (stable breakdown product of TXA ) and PGE in supernatants of
RAW 264.7 were determined by sensitive and specific ELISAs (Amer-
sham, Dübendorf, Switzerland). The assays were performed according
to the manufacturer’s instructions. Data are indicated as pg/ml.
Fig. 1. Flow cytometric analysis of RAW 264.7 cells for CD44 (A), Fcg
receptor (B), VLA-4 (C) and Thy-1.2 (D) surface expression. Gray
curve on each panel represents control staining without primary anti-
body, black areas represent positive cells. CD44, Fcg receptors, and
VLA-4 are abundantly expressed.
2
2
2

4
98
L. K. Sun et al.
Inflamm. res.
Fig. 2. RT-PCR analysis for COX-2, COX-1, PGES, and TXS in RAW 264.7 cells in response to CD44 crosslinking (A) and VLA-4 crosslinking (B).
The housekeeping gene GAPDH was also amplified for comparison. Stimulation with LPS (100 ng/ml) was used as a positive control. All four genes
are constitutively expressed. COX-2 is upregulated with CD44- and VLA-4-crosslinking and LPS, TXS with CD44-crosslinking and LPS, and PGES
with VLA-4-crosslinking and LPS.
Fig. 3. Western blot analysis for COX-2 expression in RAW 264.7 cells
in response to CD44-crosslinking with the anti-CD44 mAb
IRAWB14.4 (1–4 h), HA (100 mg/ml) and LPS (100 ng/ml) (stimulat-
ed for 2 h). All three stimuli increase the 70 kD protein. IRAWB14.4 has
a stimulatory effect also in the absence of secondary antibody. Figure is
representative of three similar experiments.
effect, as COX-2 mRNA did not increase in response to
IRAWB14.4 alone. In a control experiment, COX-2 protein
was also increased in response to VLA-4 crosslinking,
whereas crosslinking of the Fcg receptor did not enhance
COX-2 protein expression (data not shown).
Differential regulation of TXA and PGE production
2
2
We then evaluated the production of TXB and PGE after
2
2
Fig. 4. Production of TXB (A) and PGE (B) by RAW 264.7 cells.
2 2
crosslinking of CD44, VLA-4 and Fcg receptors as measures
Cells were stimulated by crosslinking with mAbs targeting the Fcg
receptor (2.4G2), CD44 (IM7.8.1 or IRAWB14.4), and VLA-4 (R1-2)
for 16 h. Controls included stimulation with LPS (100 ng/ml) and HA
of distal enzymatic events after COX-2, TXS, and PGES
induction. After crosslinking, the levels of the TXA metabo-
2
(100 mg/ml). Data are mean ± SE (n=3). Figure is representative of five
lite TXB , and PGE were assayed in the supernatants. Fig. 4
2
2
similar experiments.
demonstrates that crosslinking with the activating anti-CD44
mAb IRAWB14.4 significantly enhanced the production of
TXB , but did not stimulate the production of PGE . This was
2
2
in marked contrast with VLA-4 crosslinking, which
enhanced both TXB and PGE . TXB was only marginally
Discussion
2
2
2
increased after crosslinking of the non-activating anti-CD44
mAb IM7.8.1. Crosslinking of Fcg receptors also did not
increase TXB and PGE production. The stimulatory effect
PGs and TXs are important mediators of a wide variety of
physiological and pathological processes. PGE and TXA
2
2
2
2
of IRAWB14.4 crosslinking was comparable with LPS and
are two important eicosanoids, involved in the regulation of
HA stimulation. Interestingly, HA also stimulated PGE pro-
inflammatory and immunological events. PGE has been
2
2
duction.

Vol. 50, 2001
CD44-mediated cyclooxygenase-2 expression and thromboxane A production in RAW 264.7 macrophages
499
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shown to exhibit an anti-inflammatory and vasodilatatory
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2
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2
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2
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Acknowledgements. We thank Dr. J. Lesley for providing the
IRAWB14.4 hybridoma, and Dr. P.J. Jakobsson for sharing the mouse
PGES sequence. This study was supported by the Swiss National Sci-
ence Foundation (Grant No. 32-50721.97 to RPW), and by the Research
Commission of the Kantonsspital St. Gallen.