The Influence of Natural Antibodies on Cell Adhesion

and Migration

Petrova Yu.I., Skok M.V.

Palladin Institute of Biochemistry,

Kiev, Ukraine

• Essential amounts of pAb are present in serum of both healthy individuals and patients with autoimmune diseases and cancer

• B1 cells capable of making pAb often express T cell surface marker CD5

• CD5+ B cells comprise: 90-100% B cells in newborn up to 30% of circulating B cells in adult 10 % of B cells in spleen and 30% in Lymph nodes 30-60% of B cells in peritoneal cavity

• The function of these Ab in vivo is still unknown

• Natural or polyreactive antibodies (pAb) are naturally-occurring antibodies that can react with a variety of self and non-self antigens

The aim of present experiments was to establish

whether polyreactive antibodies (pAb) influence cell adhesion and

migration

To meet this aim monoclonal polyreactive antibody 2C3 was

generated

Monoclonal Ab 2C3 migrated in PAAG as single band and showed single band after

isoelectrofocusing

pH 5

pH 9

pH 7

Monoclonal pAb 2C3 was capable to bind structurally different antigens

0.0625 0.125 0.25 0.5 1 20.0

0.1

0.2

0.3

0.4

E492

0.0625 0.125 0.25 0.5 1 20.0

0.1

0.2

0.3

0.4

0.5

E492

0.0625 0.125 0.25 0.5 1 20.00

0.25

0.50

0.75

1.00

E492

0.0625 0.125 0.25 0.5 1 20.00

0.25

0.50

0.75

1.00

E492

0.0625 0.125 0.25 0.5 1 20.0

0.1

0.2

0.3

E492

0.0625 0.125 0.25 0.5 1 20.0

0.1

0.2

0.3

0.4

0.5

0.6

E492

[Abs], g/ml [Abs], g/ml[Abs], g/ml

D-fr of Fg

Fg

FN

ATIII

Ova

LZ

BSA

Coll I

Plg

uPA

a2-Pl

tPA

His

RNA

DNA

TG

Ins

CAM

CT

PPD

BX

pAb 2C3 was capable to bind cell antigens (immunocytochemical staining

of HT1080 cells)

Cell attachment to FN was not affected by pAb

Normal humanfibroblasts

HT1080 cells

C on tr ol

1.0

0.5

0.0

con

trol

con

trol

Ce l

l s p

e r w

ell ,

*104

pA

b

pA

b

Cell spreading on FN was not affected by pAb

Control + pAb

Cell adhesion to FN was decreased in the presence of pAb

control pAb integrin Ab0

25

50

75

100

Control IgG pAb α5β1 Ab

% o

f c e

l l a

dhe r

ed

Immunocytochemical confirmation of normal human fibroblast (А) and НТ1080 cell

(B) α5β1 expression

Immunocytochemical confirmation of normal human fibroblast (А) and НТ1080 cell

(B) α5β1 expression

А

B

Kinetics of cell adhesion to FN

0 10 20 300

1

2

3

4

Normal human

fibroblasts

HT1080 cells

0 10 20 30 40 500

1

2

3

4

Cel

ls p

er w

ell,

*104

Time, min

control

pAb

51 Ab

51 Ab

pAb

control

Phagokinetic assay was used to study cell migration

Normal human fibroblast migration was increased in the presence of

pAb

+ Control IgG

+ pAbMigration index

0.0

0.5

1.0

1.5

2.0

2.5 *

pA

b

Con

trol

HT1080 cell migration was increased in the presence of pAb

0.0

0.5

1.0

1.5

2.0 *

Control

+ pAb Migration index

pA

b

Con

trol

uPA and uPAR expression: immunocytochemical staining of

normal human fibroblasts (А) and НТ1080 cells (B)

А B

uPA

uPAR

uPA increased cell migration

control

+ uPA

Migration index0.0

0.5

1.0

1.5

2.0

2.5**

Con

trol

uPA

Both pAb and uPA increased cell migration

0.0

0.5

1.0

1.5

2.0

2.5

***

**

controlMigration index

+pAb

+uPA pAb+uPA

pAb

uPA

pAb+

uPA

Both pAb and uPA decreased cell adhesion

5.0 15.0 45.00

1

2

3

4

*

**

Time, min

ControlpAbuPApAb + uPA

PAI-1

Cel

ls p

er w

ell,

*104

In contrast to pAb, PDGF increased cell adhesion (A) and proliferation (B) but

decreased cell migration (C)

control PDGF0

1

2

3

4

5

Control

Con

trol

PD

GF

*

0.0

0.5

1.0

1.5

*

A

C

Migration index

PDGF

*

Dou

bli

ng

tim

e, h

ours

Con

trol

pA

b

B

C

ells

per

wel

l, *1

04

PD

GF

Con

trol

Summary:

• 2C3 antibody is polyreactive one and can bind cellular antigens;

• pAb 2C3 inhibits but does not block cell adhesion to ECM proteins. pAb doesn’t interfere with monovalent integrin binding. The effect is realized mainly at the adhesive complex formation stage;

• Partial inhibition by pAb 2C3 of cell-substratum adhesive interactions results in an increase of random cell migration;

• Malignant cells show higher increase of cell motility while same decrease of cell adhesion in the presence of pAb;

• The effect of pAb on cell adhesion and migration differ from that of the specific anti-integrin antibodies, PDGF or urokinase. The possible mechanism of pAb effect is a creation of steric difficulties to integrin aggregation and clustering.

CONCLUSION:

• Natural antibodies can set up an important tool for in vivo regulation of cell adhesion and motility