CD14 and Toll-like Receptors Regulate CD14 and Toll-like Receptors Regulate Lipopolysaccharide Induction of KGF-1 Lipopolysaccharide Induction of KGF-1

Protein ExpressionProtein Expression

EE Putnins*, Sanaie AR, Firth JD, Wu Q.

Faculty of Dentistry

The University of British Columbia

Vancouver, B.C., CANADA

IntroductionIntroduction Abnormal epithelial cell

proliferation is one aspect of PDD onset and progression.

Understanding the regulators of early disease onset may explain why some individuals are resistant/susceptible to disease.

If the epithelial barrier is perturbed then LPS from Gram negative microorganisms is likely to stimulate gingival fibroblasts.

Fibroblast Growth FactorsFibroblast Growth FactorsInterested in paracrine mediated epithelial

specific growth factors.

– Growth factors that are secreted by fibroblasts and specifically stimulate epithelial cell proliferation and migration.

– Keratinocyte Growth Factors-1 (FGF-7) and -2 (FGF-10).

Keratinocyte Growth FactorsKeratinocyte Growth Factors

Mitogenic for a wide variety of epithelial cells. Epithelial cells express the appropriate receptor

(FGFR2-iiib) Increased expression of KGF-1 in chronic

inflammatory conditions (e.g. Ulcerative Colitis/Crohn’s disease, Psoriasis).

What about Periodontal disease?

Gingival Fibroblast Expression of KGF-1Gingival Fibroblast Expression of KGF-1

Gingival fibroblasts express KGF-1 and 2.

KGF-1 expression was induced by: – Serum– IL-1, IL-6 and TNF-

KGF-1 expression induced by LPS purified from:– P. gingivalis (ATCC 33211)– E. coli (O55:B5)

2.4 kb

1.71.3 kb

Sanaie AR, Firth JD, Uitto V-J, and Putnins EE (2002) J Periodont Res., 37(1):66-74

Examine the signaling pathway that regulates LPS induction of KGF-1 expression.

PurposePurpose

HypothesisHypothesisLPS induction of KGF-1 protein expression is mediated

through a CD14 and Toll-like receptor signaling pathway.

Materials and MethodsMaterials and Methods Gingival fibroblasts were isolated from tissue discarded during

crown lengthening procedures. Commercially prepared E.coli LPS (O55:B5-Sigma) KGF-1 protein expression into conditioned media analyzed with

sandwich ELISA. CD14 expression was analyzed with:

– Western blotting– Immunohistochemistry– FACS

Toll-like receptors 2 and 4 expression was analyzed using FACS analysis.

Functional analysis was done using specific blocking antibodies.

LPS Induction of KGF-1 Protein LPS Induction of KGF-1 Protein ExpressionExpression

mean ±sd; n=4

KG

F-1

(pg

/ml)

LPS Induction of mCD14 on Gingival LPS Induction of mCD14 on Gingival Fibroblasts (18hr)Fibroblasts (18hr)

Control

LPS-Txd

Western Blotting

LPS stimulation of gingival fibroblasts induced a 67% induction of expression of mCD14 in total cell extracts.

LPS Induction of CD14 Membrane LPS Induction of CD14 Membrane ExpressionExpression

Induction of CD14 Expression (M1+M2).– C) control– D) 1hr (39% increase)– E) 3hr (121% increase)– F) 24hr (18% increase)

Significant increase in the number of gingival fibroblasts expressing high CD14 levels (M2 peak).

M2M1

M1 M2

M2M1

R1

M1 M2

A

D

E F

B

C

Signaling Mechanism by Which LPS Signaling Mechanism by Which LPS Regulates KGF-1 ExpressionRegulates KGF-1 Expression

Preincubation of fibroblasts with CD14 blocking antibody inhibits LPS induction of KGF-1 protein expression.

CD14 is involved in LPS signaling but it lacks a intracellular signaling tail.

mean ±sd; n=4

KG

F-1

(pg

/ml)

Gingival Fibroblast Expression of Gingival Fibroblast Expression of Toll-like Receptors 2 and 4Toll-like Receptors 2 and 4

FACS analysis Significant decrease

in cell surface expression of Toll-like receptors 2 and 4 in LPS-treated samples.

Rel

ativ

e C

han

ge in

Rec

epto

r E

xp.

Toll-like Receptor Regulates the Toll-like Receptor Regulates the LPS Induction KGF-1LPS Induction KGF-1

Preincubation of cells with Toll-like receptor-2 and -4 blocking antibodies negated LPS induction of KGF-1 protein expression.

mean ±sd; n=4

KG

F-1

(pg

/ml)

ConclusionsConclusions LPS, in a concentration dependant manner,

significantly induced KGF-1 protein expression. CD14.

– Increase in total and cell membrane expression of CD14.– CD14 is involved in the regulation of KGF-1 expression.

Toll-like receptors 2 and 4– Both are expressed on gingival fibroblasts– Rapid decrease in cell membrane expression of both

receptors is consistent with a receptor mediated internalization pathway.

– Functioning of both receptors are required for LPS induction of KGF-1 expression.

Regulation of KGF-1 Expression in Oral Chronic Regulation of KGF-1 Expression in Oral Chronic Inflammatory ConditionsInflammatory Conditions

Host Induced Host Induced ExpressionExpression

PMN

Macrophage/Monocyte

Gingival Fibroblast

IL-1TNF-

-6IL-1IL-TNF

-8IL

KGF

Junctional Epithelium

-1, -6IL IL

-6IL-1IL

LPS exp Gm neg micro

Micro Induced Micro Induced ExpressionExpression