Interleukin-1 receptor antagonist in bone marrow-derived cells

Internal Medicine I, NDMC

Interleukin-1 receptor antagonist in

bone marrow-derived cells

contributed to the suppression of

arterial inflammation and reduced

neointimal formation after injury

Sarasa Isobe1, Kikuo Isoda1, Manabu Kitagaki1 Tomiharu Niida1, Harumi Kondo1, Koji Akita1, Takehiko Kujiraoka1, Takumi Toya1, and

Takeshi Adachi1

1Internal Medicine I, National Defense Medical College

The authors have no financial conflicts

of interest to disclose concerning the

presentation.

European Society of Cardiology

COI Disclosure


Background

Interleukin (IL)-1 is a proinflammatory

cytokine that plays an important role in

inflammation. IL-1 receptor antagonist

(IL-1Ra) negatively regulates IL-1

signaling by blocking the functional

receptor. Thus, IL-1Ra plays an anti-

inflammatory role in inflammation.


Deficiency of IL-1Ra promotes neointimal

formation after cuff Injury

WT IL-1Ra-/-

H & E

E. Masson

Isoda et al. Circulation. 2003; 108: 516-518.


IL-1Ra is expressed on BM-derived cells as well

as endothelial cells at 7 days after cuff Injury

WT IL-1Ra -/-

Isoda et al. Circulation. 2003; 108: 516-518.


Study Aim

To elucidate the role of IL-1Ra

in BM-derived cells in the

development of neointimal

formation after injury.


Methods

To disrupt all four isoform of IL-1Ra gene production, a

5.2-kb DNA fragment which contains all the coding

regions for the secreted form of the IL-1Ra was deleted.

Screening of Mice: Southern blot and PCR.


Bone marrow cell transplantation

Ra-/->Ra-/-

WT >Ra-/-

WT

Ra-/-

Donor

Recipient Ra-/-

750rad


Artery injury

Polyethylene cuff was placed around the femoral artery

(reported by Moroi et al).

vein

artery

Histological analysis

After perfusion, the femoral artery was harvested, frozen in

OCT medium and sectioned.

All samples were stained with Victoria blue-HE, and

immunohistochemistry (a-SMA, PCNA, Mac3, CD31, IL-1Ra).

Imaging analysis

The lesion area was measured using NIS Element.

Chemical analysis

Enzymatic methods.


Systolic blood pressure and total

cholesterol levels in each groups

(4 weeks after BMT)

0

20

40

60

80

100

120

SB

P (

mm

Hg

)

0

20

40

60

80

100

120

TC

(mg/d

L)

Ra-/- → Ra-/- WT → Ra-/-

(n=6) (n=6) (n=6) (n=6)

Ra-/- → Ra-/- WT → Ra-/-

NS NS


The neointimal formation in BMTWT ￫ Ra-/- mice

decreased significantly compared to BMTRa-/- ￫ Ra-/- mice

100μm

WT→Ra-/- Ra-/-→Ra-/-

0

10000

20000

30000

40000

50000

60000

Ra-/- →Ra-/- WT→Ra-/- Ra-/- →Ra-/- WT→Ra-/-

0

1

2

3

4

5

I/M

Ra

tio

*** ***

*** P<0.001

(n=7) (n=8) (n=7) (n=8)


The PCNA positive cells in BMTWT ￫ Ra-/- mice

decreased significantly compared to BMTRa-/- ￫ Ra-/- mice

Ra-/-→Ra-/- WT→Ra-/-

0

20

40

60

80

100

PC

NA

po

sit

ive c

ell

s (

%)

Ra-/- →Ra-/- WT→Ra-/-

25μm

(n=6) (n=6)

***

*** P<0.001

10 days post-injury


The Mac3 (macrophage) positive area in neointima

of BMTRa-/-￫ Ra-/- mice increased significantly

compared to BMTWT￫ Ra-/- mice

50μｍ

0

5

10

15

20

Mac3 p

os

itiv

e a

rea (

%)


Ra-/- →Ra-/- WT→Ra-/- (n=6) (n=6)

***

*** P<0.001

14 days post-injury

: IEL

: Mac


The a-SMA positive area in neointima of BMTRa-/-￫ Ra-/- mice

decreased significantly compared to BMTWT￫ Ra-/- mice

0

20

40

60

80

100

SM

A p

osit

ive a

rea (

%)


Ra-/- →Ra-/- WT→Ra-/-

50μｍ

(n=6) (n=6)

** ** P<0.01

14 days post-injury

: IEL


The CD31（ endothelial cell ） positive area in neointima of

BMTRa-/-￫ Ra-/- mice decreased significantly compared to

BMTWT￫ Ra-/- mice

0

20

40

60

80

100

Re-e

nd

oth

eli

alizati

on

(%

)


Ra-/- →Ra-/- WT→Ra-/-

50μｍ

(n=6) (n=6)

*** *** P<0.001


We detected IL-1Ra protein in both inflammatory cells and

serum of BMTWT￫ Ra-/- mice, but not BMTRa-/-￫ Ra-/- mice

50μｍ

0

20

40

60

80

Seru

m I

L-1

Ra l

evel

(pg

/mL

)


Ra-/- →Ra-/- WT→Ra-/-

**** **** P<0.0001

(n=6) (n=6)

7 days post-injury

:inflammatory

cells


IL-1Ra-/- macrophages were activated excessively compared

to those from WT mice even by same levels of stimulation

Peritoneal macrophages (2 x 105 cells) were plated on a 96-well plate

and stimulated with 1mg/ml LPS for 18 hours.

0

40

80

120

WT Ra-/-

** Mac

** P<0.01


Migration activity of IL-1Ra-/- macrophages increased

compared to that of WT macrophages

Peritoneal macrophages (1 x 105 cells) were loaded into the migration chamber

and medium containing 10ng/ml MCP-1 was placed in the lower chamber. After

allowing cell migration for 12 hours, migrated cells were counted.

Mac

5000

0 WT Ra-/-

10000

15000

20000 **

** P<0.01


Neointima <<

Re-endothelialazation

>>

% a-SMA (+)

>>

Macrophage <<

Ra-/- →Ra-/- WT→Ra-/-

Summary (1) (in vivo)


Summary (2)

Cytokine

<<

Mac (Ra-/-) Mac (WT)

TNF-a TNF-a

Migration

<<

(in vitro)


Conclusion

IL-1Ra in BM-derived cells contributed to

the suppression of arterial inflammation and

the promotion of re-endothelialization,

resulting in a decrease of neointimal formation

after injury.

These novel findings may provide new

insights into the mechanisms underlying

atherosclerosis and restenosis after angioplasty.


Bone marrow cell transplantation

For BM cell transplantation, BM cells were taken

from femurs, tibias, and pelves of IL-1Ra–/– (5 to

6 weeks old) and wild-type (5 to 6 weeks old)

mice and were treated with hemolysis buffer.

Purified BM cells (107 cells/mouse) in 0.2 mL

PBS were transplanted intravenously into lethally

irradiated (750 rad) recipient mice at 4 weeks of

age (wild-type mice).

Documents

Interleukin-1 receptor antagonist in bone marrow-derived cells