The new sepsis marker, sCD14-ST (PRESEPSIN), induction mechanism in the

rabbit sepsis models

P-19

Katsuki Naitoh, Kamon Shirakawa, Jiro Hirose, Masaki Nakamura, Takashi Takeuchi, Yoshitaka Hosaka, Shoji Furusako
Discovery Research, Mochida Pharmaceutical Co., Ltd., Gotemba, Japan
Background

(1) Remarkable rise of presepsin level in CLP model, but not in LPS-induced model

9 CD14 is a high-affinity endotoxin receptor expressed on the immune cells

and an attractive target for research on infectious diseases.
9 sCD14-ST (Presepsin) is a truncated form of CD14. We generated an
anti-presepsin antibody and developed the new immunoassay to measure
a presepsin level as a diagnostic marker of sepsis and septic shock.
9 The levels of presepsin were significantly higher in the septic patients
than in the SIRS patients and healthy controls (Fig.1).
9 The production mechanism of presepsin has been investigated with in
vitro and in vivo system and our hypothesis is discussed in the poster
presentation.

3000

DiedN = 7

Rabbit prresepsin
(pg/m
mL)

: P<0.0001
(Man-Whitney test)

1500
1050

1000

19.6
(22.5)
(22
5)

723
(780)

159.0

60.5

LPS
(n=6)

Median
( interquartile ranges )
41.4
(139.4)
(139
4)

Normal
(n=75)

Sepsis
(n=55)

Control
(n=18)

CLP
(n=12)

LPS induced systemic inflammation however CLP induced

2 3).
polymicrobial infection2,3)

800
400

200

100

before 0.5

24

9 The presepsin levels were elevated in dead individuals

within 24 hr, but not in alive individuals.
9 The presepsin levels were elevated sharply prior to the
inflammation marker, IL-6.

2) Kidney International 2003;64:1620-31

3) Shock 2005;24:52-7

1) J Infect Chemother 2005;11:1234-38.

Post operation (hr)

9 Thus, it was speculated that the infectious stimulus led to

the elevation of presepsin levels.

The concentrations of presepsin were measured in plasma samples from

normal controls, septic patients, and SIRS patients by the 1st generation kit 1).

9 The presepsin level was significantly increased in the

CLP model, but not in the LPS one.

SIRS
(n=80)

AliveN = 1

1200

500
0

2000
1000

2000

MeanSE

Rabbit IL-6
(ng/mL)

Presepsin (ng/mL)

: P<0.05
(Man-Whitney test)

Fig.3 The time course of presepsin and IL-6 levels in the

CLP model.

Infection

Inflammation
2500

Rabbit presepsin (p
pg/mL)

as

Cecal ligation and puncture (CLP) sepsis model

Fig.2 Presepsin levels in two experimental sepsis models.

Fig 1 Presepsin levels were elevated in septic patients.

Fig.1
patients
4000

LPS-induced sepsis model

In vivo

(2) Secretion of presepsin in rabbit peritoneal leukocytes.

The rabbit presepsin levels in culture

supernatants were measured by ELISA.

2.0

PMA

E.Coli

LPS

0.0

Control

The peritoneal exudated cells were collected and

suspended in the assay buffer containing 2%
normal rabbit serum.

9 The presepsin level was increased specifically by

the treatment of E. Coli, demonstrating that
infection was required for the secretion of presepsin.

1.0

sC
CD14 in serum
(ng/mL)

Sepsis Serum
Normal Serum
Purified sCD14

100
0
10.0

0.0
800

6.0
0.0

Cathep
psin D
+
Pepsta
atin A

Phagolysosome

Conclusions

Purified sCD14 (non-treated)

Cathepsin D digested sCD14

400

9 10 11 12 13 14 15 16

9 Human sCD14 was purified from human serum.

Treatment of sCD14 by cathepsin D produced
presepsin over 30-fold (Fig.7).
9 The presepsin produced by cathepsin D was identical
to the fractions detected in the sepsis serum (Fig. 8).
9 An estimated molecular weight of the presepsin
produced by cathepsin D was 13kDa including Nterminal of CD14.

Phagocytosis

Lysosome

Fr. No. 5 6
a b c

9 It was speculated that the aspartic

protease cleaved CD14 to generate
i
presepsin.

CD14

Presepsin

Cathepsin D

Sepsis Serum
Normal Serum

5.0

9 An asparatic protease inhibitor,

pepstatin A suppressed the E. coli
induced presepsin secretion while an
elastase inhibitor, elaspol did not.

Phagosome
12.0

Cathep
psin D

0.0

200

25
0

Normal
Inactived
Serum
serum
(Control)

Gel filtration analysis (superdex75 column)

Presepsin (ng/mL)

2.0

C
Control

Presepsin (g/mL)

Fig.8 Analysis of CD14 derived products digested with

cathepsin D.

4.0
3.0

75
50

A putative mechanism of presepsin secretion

Purified sCD1
14
(ng/mL)

Fig.7 Generation of presepsin from sCD14 by

cathepsin D and suppression by its inhibitor.

25

9 The results indicated that phagocytosis was

involved in the secretion of presepsin .

(3) Production of presensin by the cleavage of CD14 with Cathepsin D.

Analysis of digested product with gel
filtration analysis and western blotting

50

9 The E. coli induced presepsin secretion was

suppressed by the addition of wortmannin or
cytochalasin, and also in the inactived serum.

9 The sCD14 level was elevated in LPS, E.Coli and

PMA treated groups.

In vitro
(Protein) Digestion of CD14 with a lysozomal enzyme

75

Elaspo
ol

4.0

fMLP

Peritoneal leukocytes were prepared from NZW

rabbit by injection of 0.1% glycogen.

25
0

n=3
MeanSD

6.0

50

Contrrol

Rabbit Presepsin
(ng/mL)

0.0

Preparation of rabbit peritoneal leukocytes

The peritoneal leukocytes were treated with

several stimulators (Fig.4) and inhibitors (Fig.5, 6)

100

75

% of control

Effect of protease inhibitors on presepsin secretion

125

100

% of Control

5.0

125

100

Cytochalasin
n

Effect of phagocytosis inhibitors on presepsin

secretion

10.0

125

Wortmannin
n

Induction of presepsin in rabbit peritoneal leukocyte

n=3
MeanSD

15.0

% of Control

Rabbit sCD14
(ng/mL)

20.0

Fig.6 Effects of protease inhibitors on E.

li induced
i d d presepsin
i secretion.
ti
coli

Pepstatin A

Fig.5 Effects of phagocytosis inhibitors and inactivated

serum on E.coli-induced
E li i d d presepsin
i secretion.
ti

Contro
ol

Fig.4 Secretion of sCD14 and presepsin in

peritoneal
it
l leukocytes.
l k t

In vitro
(cell assay)

Digested with Cathepsin D

4) Infect Immun. 1997; 65: 47474753

9 Presepsin markedly increased in the septic patients.

9 Microbial infection caused the elevation of the presepsin level in rabbits
model, but not LPS challenge.
9 E.Coli cells induced the secretion of presepsin from peritoneal leukocytes,
but not LPS.
LPS
9 Phagocytosis inhibitors or aspartic protease inhibitors suppressed the
secretion of presepsin in peritoneal leukocytes.
9 Human presepsin is produced by cleavage of CD14 with cathepsin D.

9Secretion of presepsin might be triggered by microbial infection.

15kDa
a : Purified sCD14 (non-treated)
b : Cathepsin D digested products
c : Molecular Weight Marker

Detection Antibody
9 Anti-CD14 N-terminal
antibody

9Phagocytosis process and cleavage with lysozomal enzymes are

involved in its secretion.
9Presepsin might be a useful biomarker for infectious diseases
including sepsis and septic shock.