Zbl. Bakt. Hyg., 1.Abt, Orig. A 250, 383-391 (1981)

Department of Veterinary Physiology, Obihiro University, Obihiro, Hokkaido, Japan

Mouse Spleen Cell-Derived Toxoplasma Growth Inhibitory Factor:

Its Effect on Toxoplasma Multiplication in the Mouse Kidney Cells *

YOSHITSUGU MATSUMOTO, HIDEYUKI NAGASAWA, HARUHISASAKURAI, SATOSHI SASAKI, and NAOYOSHI SUZUKI 1

Received February 10, 1981

Summary

When Toxoplasma tachyzoites were inoculated into normal kidney cell monolayers,they multiplied in the cells and further causing cell rupture. However, when Toxoplasmaimmune lymphokines (LKs) was added to normal kidney cells, the intracellular multi­plication of the tachyzoites was inhibited remarkably; particularly, in the case in whichthe tachyzoites were exposed to immune fresh serum prior to inoculation into the cells.

Toxoplasma growth inhibitory factor (Toxo-GIF) in LKs, with a m.w. of 30,000 to40,000 was found to be contained in LKs-II or MIF-I fraction after Sephadex G-I00 gelfiltration. LKs collected from Balb/c mice inhibited tachyzoite multiplication in ICR-JCLnormal mice kidney cells, suggestingthat LKs activity did not show mouse-strain specificity.

In contrast, the addition of Toxoplasma immune serum to infected cells had no effecton the intracellular multiplication of Toxoplasma and also did not prevent cell-to-cellspread. However, when extracellular tachyzoites were exposed to Toxoplasma immunefresh or heat-inactivated immune serum, especially to the former, at 37°C for 30 min,their active penetration into the kidney cells were extremely inhibited and their subsequentmultiplication in the cells were almost totally inhibited within 24 h as compared withthose exposed to normal serum.

Introduction

In the recent years, several kinds of Lymphokines (LKs) have been presumedto be mediators of protective immunity against obligate intracellular pathogens(2, 3, 6, 8, 10-13). Soluble mediators of immunity have been found in the super­natant of immune lymphocytes incubated with Toxoplasma specific antigen (1,2,5, 9-13). They were used with either macrophages or with monocytes. In the

" Presented at the 3rd German-Japanese Cooperative Symposium on Protozoan Dis­eases, Kyoto, Japan, Oct. 28.-29. 1980

1 This study in part was supported by Grant No. 544014 from the Scientific ResearchFund of the Japanese Ministry of Education, Science and Culture.

384 Y.Matsumoto, H.Nagasawa, H.Sakurai, S.Sasaki, and N.Suzuki

report by Chinchilla and Frenkel (3), they have identified separate and distinctmediators from hamster lymphocytes against Toxoplasma and Besnoitia. Thesemediators were effective not only for macrophages but also for kidney cells andfibroblasts.

In our previous reports (4, 9), LKs from Toxoplasma immune mice spleen cellculture with specific antigen is divided into 4 major fractions, namely: LKs-I,LKs-II, LKs-III and LKs-IV, according to the elution pattern on Sephadex G-lOOgel columns. Partially purified LKs contained 2 MIF peaks, namely: MIF-I in LKs-IIfraction and MIF-II in LKs-IV fraction. Toxo-GIF, one of the factors in LKs whichinhibits the multiplication of Toxoplasma within non-immune macrophages invitro, was detected in the LKs-II fraction with a calculated molecular weight of30,000 to 40,000. Cytotoxic substances against macrophages were observed inLKs-IV fraction, however, no Toxo-GIF was present in this fraction.

Therefore, studies on these fractions, LKs-I to LKs-IV, were done to examinewhether Toxo-GIF in LKs affects Toxoplasma multiplication in mice kidney cellmonolayers or not.

Materials and Method

Mice and Toxoplasma used: More than 500 male ICR-JCL and inbred Balb/c miceweighing 20 to 30 g were used in the experiment. They were inoculated intraperitoneallywith 100 tachyzoitcs of the S-273 strain of Toxoplasma gondii. They were challenged viathe same route with 1,000 tachyzoites of the same strain 4 wk after the first inoculation.They were further challenged with 100 tachyzoites of the RH strain after another 4 wkto obtain hyperimmune mice which will serve as donors of immune lymphocytes.

Preparation of Toxoplasma Lysate Antigen (TLA): TLA was prepared by the methoddescribed by Igarashi et a!. (4). Toxoplasma tachyzoites of the RH strain were collectedfrom the peritoneal cavity of a 3-day infected mouse and washed 3 times with Hank'sbalanced salt solution (HBSS). After washing, ten-fold sterile distilled water was added.The resulting suspension was sonicated with an ultrasonic vibrator for 3 min (100 w,Kubota Insonator, Model 200, Tokyo) and kept at 4°C for 24 h. The extracted lysateantigen was centrifuged at 10,000 rpm for 1 h. The supernatant was mixed with the samevolume of sterile 1.7% NaC!. The total protein content was estimated by the Lowry et a!.method (7) using bovine serum albumin fraction V as the standard.

Preparation of Spleen cells and lymphokines (LKs): Spleen cells were separated fromspleen suspensions of normal and Toxoplasma-infected mice by means of a slight modifica­tion of the Conray-Ficoll method (14). Cells were washed twice with heparinized HBSS.The resulting sediments were resuspended in medium TC-199 containing 20% heat­inactivated calf serum (CS) and antibiotics to a concentration of approximately 1 x 107 cellsper m!. Immune and normal spleen cells were cultured at 37°C for 48 h in a humidifiedatmosphere containing 5% CO2 in the air at a density of 1 x 107 cells/ml of mediumTC-199 with 20% CS and 50 flg/ml TLA, being the optimal concentration. Culture super­natants were pooled by centrifugation at 3,000 rpm for 30 min at 4 "C. The supernatants,hereinafter, referred to as LKs, were filtered through millipore membrane filters (0.3 fI,Type PH, Millipore Corporation, Boston, Mass.) and stored at - 80°C until use.

Preparation of Toxoplasma Immune Sera: Blood was colleetedfrom Toxoplasma hyper­immuned mice and centrifuged to obtain the serum. Antibody titers were estimated usingthe Eiken Latex Agglutination Test and were 2: 1:2048. Half of the volume of this serawas used as "Immune fresh serum" and the remaining half was inactivated at 56°C for30 min and used as "Inactivated immune sera". All test sera were diluted with TC-199cultivating medium.

Toxo-GIF Effect on Mouse Kidney Cells 385

Kidney cell monolayers : Laboratory bred healthy mice were anesthetized and the kidneyswere collected and free from fat and fibrosa. They were placed in a sterile petri dish con­taining HBSSwith penicillin (100 u per ml) and streptomycin (100,ug(ml) and then mincedwith a pair of scissors and homogenized between two slide glasses. The homogenizedtissues were transferred to a conical flask containing a Teflon-coated magnet and 40 mlof 0.25% trypsin solution was added, agitating the contents at 37°C for 40 min using amagnetic stirrer. The cells were filtered through a glass fiber column to remove largedebris and the suspension was centrifuged at 400 g (1,500 rpm) for 10 min. The sedimentwas resuspended with heparinized HBSSand washed 3 times by centrifugation at 1,500 rpmfor 10 min. The cells were suspended in TC-199 containing 20% CS and antibiotics to aconcentration of approximately 5 x 10· cells per ml of medium. One ml of the suspensionwas placed in each well of a multi dish tray (FB-16-24-TC, Linbro Chern. Co., Inc.) con­taining round coverslips and incubated in a 5% CO 2 incubator at 37°C. After 5 days,when the cells started to proliferate, nonadherent cells were removed and fresh medium wasadded. These preparative cells were reincubated for two more days until use.

Assessment of Kidney Cell Microbicidal Activity: Tachyzoites of the RH strain collectedfrom a 2-day infected mouse were exposed to either diluted or undiluted (100%) mousetoxoplasma immune fresh serum (MTIFS), mouse toxoplasma immune heat inactivatedserum (MTIHIS), mouse normal fresh serum (MNFS) or mouse normal heat inactivatedserum (MNHIS) at 37°C for 30 min in a 5% CO 2 incubator prior to inoculation into thepreviously formed kidney cell monolayers. One hour later, the supernatants were aspiratedand either TC-199 medium plus 20% CS or TC-199 medium plus LKs in 66% alone wereadded into each chamber. Incubation was continued for 48 h in a 5% C02ncubator.

On another group of kidney cell monolayers, approximately 1 x 105 tachyzoites perwell were allowed to infect the cells before the addition of LKs and observation was con­tinued for 48 h in a 5% CO 2 incubator.

The fate of the intracellular parasites was monitored by phase contrast microscopy ofthe infected cell cultures at regular intervals. Toxoplasma can be readily identified by itsmorphological appearance within cytoplasmic vacuoles, thus, at various times after Toxo­plasma infection, the coverslips were withdrawn from the trays, washed with 0.9% saline,fixed in methanol and stained with May-Grunwald Giemsa stain. The number of cellscontaining 1-5 tachyzoites, cells containing more than 6 tachyzoites and also the uninfectedcells were counted microscopically to a total of 1000 individual cells per coverslip. Thenumber of parasites per 100 cells was then calculated.

Results

Penetration of Toxoplasma tachyzoites treated with various kinds of serum onmouse kidney cells

As shown in Table 1, MTIFS, both diluted and undiluted, inhibited Toxoplasmapenetration into the kidney cells, showing 0.78 ± 0.13 and 0.66 ± 0.99 tachyzoitesper 100 cells 1 hr postinoculation. In the case of those treated with MNFS, thenumber of tachyzoites found in 100 kidney cells were 3.68 ± 0.3 in the 20 Ofodiluted MNFS and 2.28 ± 0.14 in the undiluted MNFS. On the other hand, whentachyzoites were treated with 20 Ofo MTIHIS and undiluted MTIHIS, the kidneycells contained 1.72 ± 0.18 and 1.86 ± 0.34 tachyzoites per 100 cells, respecticely.With these results, the number of tachyzoites which penetrated with kidney cellsafter treatment with either fresh or inactivated serum was definitely less than thosetreated with normal serum as shown by the minimum number of tachyzoites insidethe cells.

386 Y.Matsumoto, H.Nagasawa, H.Sakurai, S.Sasaki, and N.Suzuki

Table 1. Penetration of Toxoplasma treated with various kinds of serum diluted or un­diluted on mouse kidney cells

Treated with

Medium TC-199+ 20% mouse Toxoplasma immune fresh serum (MTIFS)+ 20% mouse Toxoplasma immune heat inactivatedserum (MTIHIS)+20% mouse Normal fresh serum (MNFS)+20% mousenormal heat inactivated serum (MNHIS)

100% mouse Toxoplasma immune fresh serum (MTIFS)100% mouse Toxoplasma immune heat inactivated serum

(MTIHIS)100% mouse normal fresh serum (MNFS)100% mouse normal heat inactivated serum (MNHIS)

"Numbers of Toxoplasmaone hour postinoculation(TP Tachyzoites/Hundredcells)

0.78 ± 0.13

1.72 ± 0.183.68 ± 0.302.90 ± 0.540.66 ± 0.07

1.86 ± 0.34

2.28 ± 0.143.30 ± 0.42

* Average numbers of Toxoplasma tachyzoites/l00 cells obtained from a total count of5000 cells per round coverslip.

Antibody titer of Toxoplasma immune serum was = 1:2048 using the Eiken latexagglutination test.

Microbicidal activities of mouse kidney cells inoculated with Toxoplasma 1 hbefore the addition of various sera

Mouse kidney cell monolayers were infected with Toxoplasma tachyzoites after­which the monolayers were washed to remove the excess parasites. Culture withdifferent media containing 20 Ofo MTIFS, MTIHIS, MNFS or MNHIS at 37 °Cwas continued for 48 h. As shown in Table 2, the mouse kidney cell monolayerscontained 0.5 ± 0.1 tachyzoites per 100 cells prior to the addition of the differentsera while after cultivation for 48 h, the total number of tachyzoites in the kidneycells were 16.2 ± 3.2 to 25.1 ± 4.1, suggesting that MTIFS did not have a directeffect on the parasite multiplication in the kidney cells.

Penetration and Multiplication of Toxoplasma treated with various kinds ofserum on mouse normal kidney cells after the addition of LKs

When toxoplasma tachyzoites were treated with MTIFS, MTIHIS, MNFS orMNHIS before inoculation into mouse normal kidney cells, the total number oftachyzoites per 100 cells were 0.7, 1.9,2.3, and 3.3, respectively, 1 h postinfections,as shown in Table 3. In groups treated with MNFS and MNHIS and cultured withTC-199 containing 20 Ofo CS for 24 and 48 h, the total number of tachyzoiteswas 25, 3 and 13, 2 after 24 hrs and 97.1 and 95.1 after 48 h, respectively,showing remarkable multiplication in the kidney cells. When the same groups,i. e. MNFS and MNHIS, were incubated with TC-199 medium containing 66 °/0LKs for 48 h, the total number of tachyzoites was 5.6 and 0.9, respectively, show­ing the remarkable inhibition of toxoplasma multiplication in the kidney cells.

Tab

le2.

Mic

rob

icid

alac

tivi

tyof

mou

seki

dney

cells

ino

cula

ted

wit

hT

oxo

pla

sma

on

eh

ou

rb

efo

reth

ead

dit

ion

ofdi

ffer

ent

sera

inth

ecu

ltu

rem

edia

Ad

dit

ion

of

oT

P

Mea

np

erce

nta

ge

ofm

ou

sek

idn

eyce

lls

wit

hT

oxop

lasm

aaf

ter

on

eh

ou

rfo

rty

-eig

ht

ho

urs

1-5

TP

~6TP

tota

lTP

OT

P1

-5T

P~6TP

:1Ic

ell

1100

cells

Icel

lto

tal

TP

1100

cell

s

TC

-19

9'm

ediu

m+

20%

MIF

S99

.5±

0.1

0.5

±0.

10

0.5

±0.

19

8.0

±0.

30.

7±

0.2

1.3

±0.

316

.2±

3.2

+20

%M

IHIS

99.5

±0.

10.

5±

0.1

00.

5±

0.1

98.1

±0.

40.

9±

0.2

1.0

±0.

216

.5±

3.5

+20

%M

NF

S99

.5±

0.1

0.5

±0.

10

0.5

±0.

197

.5±

0.4

1.3

±0.

41.

1±

0.2

20.8

±5.

4+

20%

MN

HIS

99.5

±0.

10.

5±

0.1

00.

5±

0.1

96.9

±0.

51.

0±

0.3

2.1

±0

.425

.1±

4.0

'"s; :; (1)

~ o ~ eN 00

-...j...., o >< o

Tab

le3.

Pen

etra

tio

nan

dm

ulti

plic

atio

nof

Tox

opla

sma

trea

ted

wit

hva

riou

sk

ind

sof

seru

mon

mo

use

no

rmal

kid

ney

cells

afte

rth

ead

dit

ion

0of

lym

ph

ok

ines

::;:;

t'!1 ::r:

(1) g o :; ~ o ~ (1)

Tre

ated

wit

hC

ult

ure

No

.o

fTP

Mea

np

erce

nta

ge

of

mo

use

kid

ney

cells

wit

hT

oxop

lasm

a(T

P)

afte

rm

ediu

m/1

00ce

llstw

enty

-fou

rh

ours

fort

y-e

igh

th

ou

rsT

C-1

99

Ih

r.P

.I."

OT

P1

-5T

P~6TP

tota

lT

Po

TP

1-5

TP

~6TP

tota

lT

P/c

ell

/10

0ce

lis/c

ell

/100

cells

10

0%

MT

IFS

+2

0%

CS

0.7

±0.

199

.9±

0.1

0.1

±0.

10

0.2

±0

.29

9.2

±0.

50.

7±

0.5

0.1

±0.

12.

6±

1.8

10

0%

MT

IFS

+6

6%

LK

s0.

7±

0.1

100

00

010

00

00

10

0%

MT

IHIS

+2

0%

CS

1.9

±0.

399

.8±

0.1

0.1

±0.

10.

1±

0.1

2.2

±1.

99

9.2

±0.

30.

4±

0.2

0.4

±0

.28.

5±

4.3

10

0%

MT

IHIS

+6

6%

LK

s1.

9±

0.3

99.8

±0.

10.

2±

0.1

00.

3±

0.2

99

.9±

0.1

0.1

±0.

10

0.1

±0.

1

10

0%

MN

FS

+2

0%

CS

2.3

±0.

398

.4±

0.1

0.4

±0.

31.

2±

0.2

25

.3±

4.8

89

.7±

2.6

4.4

±1.

45.

9±

1.2

97.1

±19

.31

00

%M

NF

S+

66

%L

Ks

2.3

±0.

398

.5±

0.3

1.5

±0.

30

3.3

±1.

09

8.6

±0.

31.

1±

0.3

0.3

±0

.25.

6±

1.7

10

0%

MN

HIS

+2

0%

CS

3.3

±0.

498

.7±

0.5

0.2

±0

.21.

1±

0.3

13

.2±

4.1

89.6

±2.

15.

4±

1.2

5.0

±1

.495

.1±

20

.71

00

%M

NH

IS+

66

%L

Ks

3.3

±0.

498

.8±

0.2

1.2

±0.

40

2.5

±0.

599

.5±

0.3

0.5

±0.

3°

0.9

±0.

5

•T

oxop

lasm

ata

chy

zoit

es/l

Of)

cells

wer

eo

bta

ined

fro

ma

tota

lco

un

to

f50

00ce

llsp

erro

un

dco

vers

lip.

Ta

ble

4.M

icro

bic

idal

acti

viti

esof

ICR

-JC

Ln

orm

alm

ouse

kid

ney

cells

inoc

ulat

edw

ith

To

xopl

asm

as(T

p)on

eh

ou

rb

efor

eth

ead

dit

ion

cfT

oxo

plas

ma

imm

un

eIC

R-J

CL

mou

seIy

mph

okin

es(L

Ks)

,MIF

-Ia

ndM

IF-I

Iin

the

LK

s

Vol

00

00

Rem

arks

:M

ediu

mT

C-1

99+

20%

CS,

med

ium

cont

aini

ng

twen

type

rcen

tca

lfse

rum

.

Tab

le5.

Mic

rob

icid

alac

tivi

ties

ofI

CR

-JC

Ln

orm

alm

ouse

kid

ney

cells

ino

cula

ted

wit

hT

oxo

plas

ma

(Tp

)o

ne

ho

ur

bef

ore

the

add

itio

no

fT

ox

o­

plas

ma

imm

un

eR

aib

lem

ouse

lyrn

phok

ines

(LK

s),

MIF

-Ian

dM

IF-l

lin

the

LK

s

Add

itio

no

fM

ean

per

cen

tage

of

ICR

-]C

Lm

ous

eki

dn

eyce

lls

wit

hT

oxop

lasm

aaf

ter

on

eh

ou

rtw

enty

-fo

ur

ho

urs

fort

y-ei

ght

ho

urs

oT

P1-

5T

P~6TP

tota

lT

PO

TP

1-5

TP

~6TP

tota

lT

PO

TP

1 -5

TP

~6

TP

tota

lT

PIc

ell

1100

cell

sIc

ell

/10

0ce

llsIc

ell

/l0

0ce

lls

To

tal

LK

s98

.9±

0.4

1.1

±0.

30

1.1

±0

,499

.1±

0.2

OJl

±0.

20.

1±

0.1

2.8

±0

.896

.7±

1.1

2.6

±0

.90

.7±

0.3

12.2

±3.

9!l

.lIF-

I(L

Ks-

lI)

98.9

±0.

41.

1±

0.3

01.

1±

0,4

98.8

±0

.30

.9±

0.2

0.3

±0.

14

.0±

1.3

98.6

±0

.61.

0±

0.4

0.4

±0

.25.

0±

2.5

MIF

-II(

LKs-

IV)

98.9

±0.

41.

1±

0.3

01.

1±

0.4

96.9

±0

.91.

5±

0.5

1.6

±0.

533

.6±

0.7

87.1

±2.

55.

5±

1.1

6.0

±1.

111

3.5

±21

.7M

ediu

m19

998

.9±

0.4

1.1

±0.

30

1.1

±0.

498

.5±

0.4

0.7

±0.

30.

9±

0.1

15.5

±4.

489

.9±

1.9

4.2

±0.

85

.9±

1.2

96.5

±20

.7+

20%

CS

'

Mea

npe

rcen

tage

of

mou

sek

idn

eyce

llsw

ith

TP

afte

ron

eh

our

fort

y-ei

gh

th

ours

1-5

TP

~6

TP

tota

lT

P0

TP

1-5

TP

~6

TP

IceH

110

0ce

lls

Icel

lll> :l I:

l. Z en c N c :r.:-< ~ ~ In C 3 o .? ;r: Z ll> (IQ ll> In ll> :;: Jl ;r: en ll> :>

;'C ,.., !=.

:" V> ll> In ll' "'"

tota

lT

P11

00

cell

s

19.4

±3

.511

.0±

3.9

111.

5±

19.3

124.

4±

22.0

1.2

±0.

30.

9±

0.4

6.4

±0.

97.

5±

1.3

1.7

±0.

51.

3±

0.4

5.5

±1.

04.

4±

0.8

97.1

±0.

597

.8±

0.6

88.1

±1.

488

.1±

1.8

2.3

±0

.6o

2.3

±0

.6

oT

P

97.7

±0.

6

Add

itio

no

f

Tot

alL

Ks

MIF

-I(L

Ks-

lI)

MIF

-ll

(LK

s-IV

)M

ediu

m19

9+

20%

CS"

Rem

arks

:"m

ediu

mco

nta

inin

gtw

enty

perc

ent

heat

inac

tivat

edca

lfse

rum

.

Toxo-GIF Effect on Mouse Kidney Cells 389

In groups treated with MTIFS and MTIHIS and incubated with TC-199 me­dium with 20 Ofo CS alone, the total numb er of tachyzoites in the kidn ey cells was0.2 ± 0.2 in MTIFS and 2.2 ± MTIHIS after 24 h and 2.6 ± 1.8 in MTIFS and8.5 ± 4.3 in MTIHIS after 48 h, showi ng a slight tendency to increase in multipli­cation. Ho wever, when they were incub ated with TC-1 99 medium containing66 Ofo LKs for 24 and 48 h, no rachyzoites were found in the kidney cells withtho se treated with MTIFS and only 0.3 with those treated with MTIHIS after 24 h.Likewise, the same result was obtained with rachyzoites treated with MTIFS andonly 0.1 with those treated with MTIHIS after 48 h. These show that To xoplasmamultiplication was almos t completely inhibited in these groups. In all groups whereLKs were added in the culture medium for 24 and 48 h, T oxoplasma multiplicationin the kidney cells was inhibited remarkably as compared with those groups inwhich LKs was not added at all.

Microbicidal activity of ICR -JCL normal mi ce kidn ey cells inoculated withToxoplasma 1 h before the addition of aut ologous LKs, MIF-I (in LKs-ll) andMIF-ll (in LKs-IV )

As shown in Table 4, LKs-II which also cont ains MIF-I, inhibited To x oplasmamultiplication remarkable in the kidney cell monolayers, show ing 5.0 ± 2.5 totalparasites in 100 cells after 48 h incubation. The act ivity of LKs-II to inhib it T oxo­plasma multiplication in the kidney cells was almost equa l to that of total LKs.No inhib itory effect in T oxoplasma multiplication in the cells was found whenLKs-IV containing MIF-II was added. No te that Toxoplasma organisms multipliedcomp arabl y with those grou ps in which medium TC-199 containing 20 010 CS alonewas added. In this expe riment, therefore, LKs-II contained Toxo-GIF thu s inhib­ited T oxoplasma multiplication in the kidne y cells, however, it was not cont ainedin LKs-IV.

Mic robicidal activities of ICR-JCL normal mouse kidney cells inoculated toitbToxoplasma 1 h before the addition of To xoplasma immune Ba1blc LKs, MIF-I(in LKs-ll ) and M IF-JI (in LKs-IV )

As shown in Table 5, LKs obtained from the immune spleen cells of Balblcmice were examined for its inhibito ry effect on Toxoplasma multiplication in micekidney cells collected from ICR-]CL mouse strain. When total LKs and LKs-1Ifrom Balblc mice were adde d to the ICR-]CL mice kidney cell monolayers, inhibi­tion of Toxoplasma multiplication in the cells was definitely observed, showing19.4 ± 3.5 and 11.0 ±3.9 tachyzoites per 100 cells compared to those groupswherein only TC-199 with 20 010 CS without LKs alone was added. In the latter,a total of 24.4 ± 22.0 tachyzoites per 100 cells were found.

Discussion

Previous studies proved that non-immune mouse macroph ages obtained fro mBalblc strain or ICR-JCL str ain, upon culture with LKs obtained from T oxo­plasma hyperimmuned spleen cells incubated with specific ant igen, and wh ichwhen infected with T ox oplasma tachyzoites, could show a significant inhib itionof intracellular multiplication of the organisms (4, 10, 12). Upon separation of the

26 Zbl. Bakt. H yg. , I. Abt. O rig. A 250

390 Y.Matsumoto, H .Nagasawa, H.Sakurai , S.Sa saki, and N.Suzuki

LKs into four fractions , LKs-I, LKs-II, LKs-III and LKs-IV by Sephadex G-lOO gelfiltration or Sacucynyl G-lOO gel filtration, Toxo-GIF was found to be present inLKs-II in which MIF-I was also detected with a molecular weight of 30,000 to40,000. LKs-IV which contained MIF-II, with a calculated molecular weight of3,000 to 5,000 (9), did not contain Toxo-GIF.

In this experiment, the inhibitor y effect of LKs, LKs-II and LKs-IV 00 T oxo­plasma multiplication was observed not in macrophages but in kidney cells fromICR-JCL and Ba1b/c mice. In the results, when To xoplasma tachyzoites weretreated with To xoplasma immune fresh serum at 37 °C for 30 min, tachyzoitepenetration into the kidney cell monolayers was definitely inhibited comp aredwith those treated with normal serum. Since Toxoplasma multiplication was ob­served in kidney cells when infected 1 h prior to the addition of Toxoplasma im­mune fresh serum after 48 h, we therefore conclude that Toxoplasma immunefresh serum does not have a direct effectwhen the organisms are already intracellular.Based on our results, the most perfect inhibitory effect could be obtained with theuse of LKs-II or total LKs combined with immune fresh serum. These findings onLKs-II or Toxo-GIF arc consistent with previou sly observed results using normalperitoneal macrophages, suggesting their possible identicality (9). The activity ofToxo-GIF is not mouse-strain specific and this was also observed in our results.LKs in mice therefore could confer inhibition not on ly in strains of mice fromwhich it was der ived but also in some other mice strains.

References

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Toxo-GIF Effect on Mouse Kidney Cells 391

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13. Suzuki, N ., Y.Hirota, Y.li;ima, S.Makimura, I. Tomoda, and T.lshii: Studies on im­munoglobulins from normal and Toxo plasma infected rat sera on DEAE sephadex A-50in multilayer microcolumn s. l ap. J. vet. Sci. 45 (1975) 279-287

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Prof . Dr. N.Suzuki, Dept. of Vet. Physiology, Obihiro University, Obihiro, Hokkaido,japan