Attenuation of Murine Lupus Nephritis by Mycophenolate Mofetil

J Am

Attenuation Mofetil

of Murine

MIEKE

C. J. VAN H. M. BERDEN

Jo Division

Abstract.

Mycophenolate

(MMF)

of mycophenolic acid, is effective in prolonging

xenografts.

However,

mechanism

this

study,

the

progression

med.

effect

in the

with

day.

animals of

by

controls

(cumulative

glomerulonephritis

treated

mice

than

and

less

in the

sive

that with

and

clinical

relatively

hydrolyzed

low

by

esterases acid

type

2 isoform

of inosine

sine

monophosphate of de

(MPA).

This

surface

T and B cells, possible

via

(1-4).

since the

MMF

in other

salvage

in signifiin

it has

purine

pathway,

which

of immune

albografts,

but

(reviewed

in references

in inhiof

for

is still

inflicted

by

in Lewis

delayed

occurrence

of acute

rejection

of organ

deterioration

In the effect

of MMF

of action.

December

22, to Dr.

University

Hospital

“St.

1997.

Accepted

Mieke

February

C. J. van

Radboud,”

P.O.

Bruggen, Box

9101,

17,

1998.

Division

both

6500

tis

Journal

Copyright

The

by the

Society American

Society

of Nephmology

to

In

ously

we

to

renal to

perfusion a

decreased

capillary

treat

ongoing

is less

wall

rejection

experience

MMF

was

prevent

the

NZB/W

mice,

function

and

with found

autoimmune

has

in vivo

to

uveoreti-

development

of

MMF

treatment

prolonged

survival

on

the

main

mice

the

mechanism

antibody

formation

of the important

features.

From

an ultimately the

glomerular is instrumental

study,

of

we assessed

MMF

MRL/lpr

terms

on

mouse

disease, of

role

clinical,

immune

(12). binding

basement

For of

for

progresspontane-

which

closely

histologic,

and

the age of 12 to 14 wk, these fatal

gbomerulonephritis the

influence

autoimmune in

glomerulonephritis,

In this

the

on

because

This

SLE

develop

to

in MRL/bpr and

effects

and

(5)

a systemic

human

proliferative

evaluated

progression

MMF

focused

develops

to

study,

glomerubonephritis.

nucleosomes,

of Nephrology

the

in systemic lupus erythematosus (SLE) of tissue lesions, especially gbomerulonephri-

we

of

bodies

l407$03.00/0

of the American tO 1998

Nijmegen,

in MRL/ immunoglob-

beads

diseases.

of renal

and

(10,11),

this

Netherlands. 1046-6673/0908-

HB

Nephrology,

and

used

and

(8).

on disease

in vitro

start of

be

(7)

Because

immunologic Correspondence

albuminuria

1 and 6). There

rats

present

resembles Received

basement

mice compared with suppresses the devel-

(9).

sion

the

antibody

glomerubar

of experimental

rats

in BB

in vivo

effect.

in BALB/c

of glomerubar

autoimmune

diabetes

of cell

specificity not

nitis

primary

in the gbomerular

also

development

of autoantibodies the development

reduces

it can

the

mo-

found

treatment

complexes

not

no immunosup-

albumin. Interestingly, that binding of nucleo-

mice

MMF

CD8

were

nephritis.

MMF. The insights in the mechanism of action of MMF are mainly based on in vitro studies (4,5), and it remains to be determined whether similar mechanisms are responsible for the MMF

that

and

and

and

reduction

and Also,

blood

were

to the

in MMF-treated

binding in lupus

CD4,

hypersensitivity

glomerulonephritis

indicate

inhibit

synthesis is

deposits

to

of the

CD3,

MMF

observed

MMF-treated formation.

Furthermore,

of 90 mg/kg

studies

blood

of glycosylation

groups.

observed

and

results

the

ulin

The

in treating

in reduction

a relative

of bupus

MMF

thereby

has

mice.

opment

is

the action

both

of

complexes

control

and

were

and peripheral

is decreased in MMF-treated It is concluded that MMF

MMF

dehydrogenase.

cells

been

effects

response to methylated bovine serum renal perfusion experiments revealed

orally,

liver,

and inhibition

glycoproteins

was

nodes,

delayed-type

lpr mice.

is immunosuppres-

blocks

and

in

Surprisingly,

inhibition

synthesis

T and B cell proliferation

wall

numbers

properties on

membrane

in MMF-

Furthermore,

and

lymph

some/antinucleosome

wk

immunoglobulin

intestine, MPA

The

0.0001).

of

monophosphate

purine

23 =

0.005).

(MMF)

dehydrogenase

novo

at

P

Administered

in the

mycophenolic

bition

toxicity.

17).

=

a

significantly

transplantation,

mofetil

once

vehicle-treated

0.002).

organ

mycophenolate

(n was

capillary (P

mice

mg/kg)

less severe

amount

glomerular

between

mice

with

=

different pressive

in controls;

(P

the

in MMF-treated

In experimental shown

mice

studies

disease

orally

albuminuria

88%

in spleen,

exam-

h)

histologically

in control

C3 deposits

cantly

of

versus

enlargement

was

alone

pg/l8

spleen

were

1998

Netherlands.

there were no differences between animals with regard to autoantibody

18)

(90

P. M. RIJKE,

immune-modulating

T cells

In

The

no clear-cut

because control

the

TRUUS

Nzjmegen,

of lupus =

compared

was

immunofluorescence

(n

vehicle

incidence

22%

and

diseases.

spontaneous

model

(>300

treatment

mice;

the

mice

received

MMF

MMF-treated

on

WALGREEN,

vivo

9: 1407-1415.

by Mycophenolate

St. Radboud,

morpholin-

the effects

in a vehicle

albuminuria

reduced

about

mouse

dissolved

the

in autoimmune

MMF

MRL/lpr

MMF

Control

incidence

is known

MRL/Ipr

Hospital

which is its active metabsurvival of albografts and

of MMF

of

Eight-week-old

treated

The

little

of action

is

Nephritis

BIRGITTE

University

mofetil

oethyb ester olite. MMF main

BRUGGEN,

Nephrology,

of

Lupus

Soc Nephrol

mice

complex-mediated

the development antinuclear

membrane

(GBM),

of MMF

on proteinuria,

(13-15).

the effect

of

autoanti-

via

1408

Journal

on histologic merubar

lpr

American

severity

addition,

after

both

prophylactic

we

analyzed

the

assessing

the

evaluated

effect

spleen/lymph

node

and

and

main

of

toward

Furthermore,

on

in t’ivo

immune

complexes in predisease

and

serum

and

p=0.0001 80

a)

E 60

C

the

E 40 .0

antibody

(61

(mBSA).

on the binding in a renal

20

of

perfusion

mice. 13

Materials

and

Animals MRL/bpr the University Scripps Clinic Jackson immune

mice

were bred

in the animal

from stock Foundation

(Bar Harbor, ME). by MMF, BALB/c

the Jackson

Laboratory)

and

facilities

originally obtained (La Jolla, CA) For the in vivo mice were used

bred

in our

of

via from

the the

assessment

(also

central

of

obtained

animal

1. Cumulative

mg/kg

body

exceeded

labora-

ter,

a prodrug

Ltd.,

sodium

of MPA,

United

Kingdom)

chloride

polysorbate This

For

the

MMF,

first

set

8-wk-old

mg/kg

body

were

(ii

from

1 7) received

during levels

treatment, blood was drawn toward DNA, nucleosome,

week

The

“surrogate”). nucleosome/Ig by urine

weight)

and

was

considered

300

collection the

for histology.

animals

were

In a second

MMF

on established

ment,

2+).

treated

mild

with

gb(score

J Am

Soc

Nephrol

Figure

3. Glomerular

In addition

tis.

9: 1407-1415,

nuclear

cell

histology.

to this influx

to 3+ scale. some/moderate

This

of the

Deposition incubating

gbomerubonephritis

scale influx;

in

was

microscopy to study

Technika,

the degree

assessed

was

and

performed

deposition

of gbomeruli

of mono-

scored

on a 0

1 + to 2+,

on 2-p.m

of mouse

of mouse

goat

lands)

I :40.

diluted

After

Ig and

cryostat

mouse

(Nordic,

by Ig

diluted

(wt/vol)

BSA.

The

The BALB/c

with

Nether-

United Blinded

investigators. and

The

the mesangium

in at beast

Assessment

25

were

embedded

Kingdom) sections

intensity was

were

per

with

examined

of the

scored

glomerubi

in Aqua-

and examined

by

staining

in the

semiquantitativeby

on

mouse

kidney.

of Immune-Modulating

MRL/lpr

primary antibody (19). In brief, mg/kg

mice

of

adjuvant

ear.

As

left

ear.

mice

S p.1 of

with

17, blood

the anti-mBSA

Vitro

saline

micrometer.

from

on Spleen

day

was given

was

The

of the right

injected

thickness

DTH

48 h after

the

(90

0 until

same

reactivity

is

site

(R)

the rechablenge.

animals

to determine

immunosorbent

Cell

in the

( X I0)

of the antigen-injected

in enzyme-linked

of MMF

of MMF day

the pinna

of ear

site (L) as measured

Effect

previously groups of

in 0. 1 ml of complete

into

measurements

collected

response

dose from

(B).

in normal DTH and

15, a rechablenge

(2 mg/mb)

of the swelling

was

described in two

0. 1 mg of mBSA

an engineer’s

as the ratio

or MMF

also studied of MMF on

a daily

At day

1409

(A)

of vehicle

phosphate-buffered

At 48 h, duplicate

At day

vehicle

received volume

received

and the saline-injected

J

that

subcutaneously

control,

made

with

Mofetil

properties were the influence

subcutaneously.

5 p.1 of mBSA

were

treated

or an equivalent

I 7. At day 4, all mice Freund’s

by Mycophenolate

response according to methods DTH reactivity was determined

BALB/c

orally)

MRUlpr The

Properties

of Lupus

immunosuppressive mice by measuring

lO-wk-obd

C3.

the sections

Tilburg,

the sections

Poole, microscope.

scale

In Vivo

C3c

procedure,

Chemicals,

loops

1%

by incubating

from

expressed

independent

a 0 to 4+

10 mg/mI

containing

studied

anti-mouse

fluorescence

capillary

Belgium)

saline

C3 was

the staining (BDH

a Zeiss

Turnhout,

phosphate-buffered

FITC-babeled

three

score,

loops

was defined as follows: 0, no influx; and 2+ to 3+, extensive influx.

kidneys

Organon

Deposition

mount

photomicrographs

of mouse Ig was studied in direct immunofluorescence the sections with FITC-labebed F(ab), sheep anti-mouse

(Cappeb, I :750

Representative

in capillary

Immunofluorescence sections

Attenuation

1998

assay

Proliferation

(19).

of

Mice in vitro

stimulated

effect

of MMF

mitogenesis

was

of spleen

tested

on concanavalin

cells.

Spleen

cells

A (ConA)from

MRLIIpr

of MMF In the plasma the

samples

Ig concentrations

timatrigel

(anti-GBM)

immunosorbent someflg

of MMF and

the

or vehicle-treated

anti-DNA,

reactivities

assay,

complex

as

assay

were

determined

described was

MRL/bpr

antinucleosome, before

performed

an-

in enzyme-linked (13,14).

as

mice, and

The

described

nucleo-

--

previously

(14,18).

2

The effect number

of

numbers spleen,

of MMF leukocytes

on spleen

in blood,

MRL/lpr (90 mg/kg)

FITC-conjugated thiocyanate-conjugated jugated

rat

anti-mouse

for 30 mm. .

conjugated

rabbit

cells

washed

were

CD8a

hamster .

and

on

T cells in studied in 8-

**

(with

*

tetramethybrhodamine isoCD3 or with FITC-con-

CD3

was

Ig (DAKO, times

nodes,

tetramethybrhodamine

for B cells

.

three

CD4 and anti-mouse and

anti-mouse

lymph

on

for 4 wk (,z = 6) and 7 wk (n = 3) Cells (5 X l0) were incubated with

anti-mouse

Staining

and

node weight,

CD8, and CD4/CD8 peripheral blood was

mice treated or vehicle.

rat anti-mouse hamster

nate-conjugated

and lymph

spleen,

of B cells, CD3, CD4, axilbary lymph nodes, and

to 9-wk-old with MMF

CA)

treatment

isothiocya-

o

(Pharmingen,

San

Diego,

performed

using

FITC-

Denmark).

The

Gbostrup,

phosphate-buffered

saline,

Ig

glomerular 4. The amount loops in gbomerubi of significant decrease in in MMF-treated mice

I%

Figure

BSA, pH 7.4) and analyzed using a Coulter Epics XL flow cytometer (Coubter, Hialeah, FL). The percentage of CD4/CD8 T cells was calculated as follows: %(CD4/CD8) = %(CD3 ) [%(CD3/ CD4) + %(CD3/CD8)].

0.0017;

-

C3

**

0.0002.

deposits

.

of Ig and C3 deposits in glomerular capillary mice treated with MMF () or vehicle (0). A the amount of Ig and C3 deposits was observed compared with vehicle-treated controls. *P

1410

Journal

5. Representative

Figure

treatment

mice

(n

3; age,

=

l0

cells/well

7 to 8 wk)

findings

incubated

(Sigma)

concentration

MRL/lpr

for 72 h at 37#{176}C in a 5%

in a dose

(mg/mb) and

mice

mycophenobate

of I .25 p.g/mb.

and

and

mofetil

and Treatment (wk)

Prolifer-

after

(MMF)

titers

in plasma

treatment

with

of

MMF

3.7

±

1.2

3.2

±

1.0

4.2

±

1.3

4.1

±

1.1

6

6.6±2.6

5.9±2.1

9

8.9

8.9

20.0

4.7

± 5.1

19.1

0

1365

±

3

4471

± 651

6 9

7059 10,773

±

1358

±

12

12,141

± 4.7 ± 6.1

109

1555

±

147

5155

±

1346

6267

± 887

2198

8355

±

1517

± 2212

15,555

±

2212

in

Control

0

644±50 2400 ± 368

788±136

3

2071

± 254

6 9

5918 6035

±

577

4222

± 701

±

1115

7688

±

12

7764

±

1909

13,244

2680

± 3264

Antimatrigel

0

72±8

92±8

3

264±34

187±22

6 9

321±31 400±50

276±40 316±45

12

413±83

390±54

for

any

±

SD. There

parameter

were

at any

no statistically

time

assessed

deposition

1407-1415,

1998

of Ig after

vehicle

by incorporation during

of tritiated

the last

MRL/lpr a vehicle

mice (90

mice

renal

(n

4) were

=

mg/kg

(n

study,

treated

wt),

4) received

=

perfusion

body

once

equal

the

for

Blinded The

point.

antibody

mAb

34 were

was performed as described kidney was taken out and

nitrogen

evaluation

sections

amount

mesangium

(1

of

were

and

intensity

were

scored

with

MMF

a day,

for

5 wk.

were

of vehicle

anesthetized

by

(Narcovet, Apantibodies of the

obtained

by purification

of

as described antinucleosome

(13). an-

before (13). After perfusion, the immediately snap-frozen in liquid

IgG2a

examined

orally amounts

mice

supematant under physiologic conditions, perfusion of these nucleosome-complexed

tibodies perfused

thymidine

I 8 h of incubation.

of Nucleosome-Complexed Antibody

MRL/lpr For

binding

by

by three

immunofluorescence.

independent

investigators.

of the staining

in the capillary

semiquantitativeby

on

loops

a 0 to 3+

and the scale,

as

follows: 0, no staining; 1 +, mild staining; 2+, moderate 3+, strong staining. For the staining of GBM-heparan

staining; and sulfate (HS),

indirect

to techniques

immunofluorescence

described

previously

Statistical

Antinucleosome

as mean

showing

concentration)

antinucleosomal

3

are given

mice

intraperitoneal administration of sodium pentobarbitab pharma, Arnhem, The Netherlands). Complexed

concentration

differences

dissolved

culture Renal

±

was

final

alone.

or vehiclea

Vehicle

in wells

p.Ci/mb,

Six-week-old

0

Results

of MRLIbpr

Renal Perfusion Antinucleosome

Anti-DNA

a

ation

anti-DNA,

antimatrigel

before

12

significant

of glomerubi

9:

(B).

were

antinucleosome,

IgG

J Am Soc Nephrol

of Nephrology

of the immunofluorescence

treatment

by ConA

1. IgG

Parameter

Society

in 96-well microtiter plates at a concentration of 5 X in the presence or absence of MMF (0 to S p.M). Cells

stimulated

Table

American

examples

(A) or MMF

CO, atmosphere were

of the

was

performed,

according

(17).

Analyses

Statistical

analyses

were

performed

using

the

Mann-Whitney

U

test.

Results Effects

of MMF

on

Mice: Attenuation of Nephritis In the significantly vehicle-treated

preventive

the

Disease

was

whereas

the

the administration

and

dose

of 90

of abbuminuria

0.0001)

of albuminuria in

in MRU1pr

Symptoms

a daily onset

(P

controls

incidence

Furthermore,

study,

reduced

cumulative 88%,

Glomerular of Clinical

(Figure

Severity mg/kg

MMF

compared

with

1). At 23 wk,

in vehicle-treated

MMF-treated

of MMF

group

significantly

the

animals it

was

22%.

reduced

J Am

Soc Nephrol

Table

2.

9: 1407-1415,

Effects

after

Attenuation

1998

4 wk (n

6) and

7 wk

3) of treatment

(n

of MRLIlpr

of Lupus

mice

by Mycophenolate

with

MMF

Mofetil

or vehicbea

Wee k 4

Wee k 7

Parameter Vehicle Weight

MMF

Vehicle

MMF

(mg)

spleen

170 ± 20

lymph node

57

190 ± 31

3

±

57 ±

265

10

± 99

69 ±

255

10

50

±

65 ± 9

Leukocytes spleen

X

lymph

node

l0

blood

X

13.1 X

i0

106/mb

15.0

± 3.2

12.3

±

10

17.0

± 7.1

3.3

± 4.6 ±

1.0

4.4

± 3.8

4.1

±

1.9

4.2

± 3.1

2.5

±

1.5

2.1

±

2.2

±

1.7

1.7

±

36.1

±

10

41.0±11

30.3

± 9.1

1.5

1.4

% B cells spleen

41.2±

lymph

node

26.5

blood

lymph

± 4.8

5.5

10.8

± 8.7

13.3

1.8 ± 2.1

lymph

node

blood % CD8

node

blood CD4/CD8

significant

differences

MMF-

and

(Figures control

2 and MRL/bpr

of the

18 mice

were

14.3

±

6.2

10

29.0

±

Il

±

2.1

4.0

± 2.3

1.8 ± 2.1

4.0

20.8

± 6.4

19.0

± 9.1

17.0

± 6.9

34.3

± 5.1

31.2

± 9.9

27.7

± 10

29.7

± 4.1

32.3

± 2.9

25.3

± 6.8

30.0

± 3.5

± 4.2

7.8

± 6.1

4.1

20.2

±

20.9

± 3.3

9.8

± 2.1

9.3

± 1.4

8.8

23.0

± 5.7

23.8

± 5.6

18.1

±

17.7

± 4.7

21.8

± 2.9

16.8

± 5.0

2.2

± 0.7

2.3

± 0.6

2.1

± 0.9

mice,

treated

MMF

with

the

for

1 .4 ± 0.4

1.7 ± 0.4

1.5

1.5 ± 0.6

1.4 ± 0.3

2.4

0.005)

=

(P