Interação entre Trypanosoma cruzi e macrófagos: diferenças entre tripomastigotas sangüí-colas e de cultivo de tecidos.

Rev. Inst. Med. trop. São paulo 26(4):179-185, julho-agosto, 1984

TRYPANOSOMA ORUZI INTERAGT|ON

wrrH

MAGROPHAGES: D|FFERENGES BETWEEN TISSUE CULTURE AND BLOODSTREAM FORMS (")

Judiúh K. KLOETZEL (1), Regina V. MILDER (2,) and Eufrosina S. UMEZAWA (r)

sulv{MARY

i

Mouse peritoneal elicited macrophages cultured. on coverslips .were infected

with

rr¡4ranosoma cruzi trypomastigotes

from

looth the

F

strain and

y

strain obtained either

from

tissue culture

or

from

the bloodstream

_of

infected. mice.

Both the Y strain and F parasites obtained from tissue culture were interiorized

by

macrophages

at a

much

higher rate than

blood.stream tqypomastigotes.

Tissue culture parasites incubated

\¡/ith

normal mouse serum, mouse plasma

obtained at the zth day after infection,

_or

specific r4'yperimmune serum

at

sub_ agglutinating concentration, behaved. essentially as non-opsonized. parasites. ul_

frastructural differences were seen

at

the early interaction phase bet.ween ma_ crophages and trypimastigotes

from

both sources. After

:ì0 minutes, tissue

cult'ure t44pomastigotes

_were

located

in

clusters

_{at the}

area

of

contact

with macrophages. \Mhile bloodstream trypomastigotes, at B hours post-infection

were most frequently enclosed

in

a loose phagocytic vacuole, tissue culture try_ pomastigotes were enclosed

in

single

tight

vacuoles.

Both

tissue culture and

bloodstream trypomastigotes

_of

the

y

strain multiplied. 'within macrophages; F

strain bloodstream trypornastigotes did not develop within the host cells. white tissue culture trypomastigotes multiplied.

UDC 593 .16t.73

616.937.3-092.9

Several papers have been pulolished on the

interaction between T. cruzi and macrophages

with

both epimastigotes and. tr.ypomastigotes,

the latter

being

the

forms able

to

multiply

within

mammalian cells

z. In

most

of

these

papers t4ypomastigotes

are

either

obtained

from

the bloodstream

of

infected animals or

from

acellular

cultures.

Bloodstream trypo-mastigotes tend

to

infect only a small percen-tage of macrophages in vitro, ,while _{the uptake}

of

parasites obtained

from

acellular cultures

is

more intense 2.

An

alternative source

of

trypomastigotes

are infected tissue cultures; these forms have

INTRODUCTION

(1) rnstituto de Medicina Tropical de são paulo, _{university of seo paulo school of}

Medicine

(2) Instituto de Medicina Tropical de São paulo, University of Sáo paulo School

sitology

(") This 'work was supported þy cNPq Grants 2222.8.09?/80 and 2222.8.141/80 and FINEP crant 8?680/23100/00

Address: Âv. Dr. Enéias C. Aguiar 4?0, 05408 São pautò, Brasit

been rarely employed in studies on the

interac-tion between tr1¡Oanosomes and macrophages.

They are reported as being either destroyed

within these cells s

or

surviving and multiply-ing Ín their interior i5.

Recently

a

study comparing macrophage

interaction

with

bloodstream, tissue culture

and acellular culture derived tr)æomastigotes

of

the

Y

and CL strains has þeen published.

Tissue culture parasites

of

both

strains are incorporated. more intensely than bloodstream

forms, and parasites of both sources multiply

within these cells. However, CL strain parasi-Medicine, Department of Preventive

Pata-KLOETZEL, J. K.; MILDER,, R. V. & UMEZAWA, E. S. _- Trypanosoma cruzi interaction with macrophages: Differences

between tissue culture and bloodstl'eam forms. Rev, Inst. Med. úrop, São Paulo 26:U9-185, 1984.

tes

of

both sources infect macrophages

to

â lesser extent than their Y strain counterpartsrJ.

In

the present paper bloodstream and

tis-sue culture t{ypomastigotes

of

the

Y

and 1¡'

strains interacting

with

mouse peritoneal ma-crophages

"in

vitro",

v/ere compared.

An

at-tempt ,was _{also made to correlate quantitative}

differences

of

parasite uptake

with

the pres-ence of host serur¡r components on

t{ypanoso-me's membrane.

At

the

ultrastructural levei,

comparison between bloodstream and tissue

culture trypomastigotes

at

the

initial

interac-tion'with macrophages 'was made

for

the first

time, showing visible differences.

MATERIAL AND METHODS

Parasites

_-

The

Y

strain ró _and

_F

_strain 4

of T. cruzi were employed.

Y

strain is

reticu-lotropic and r4yotropic, and has characteristi-calfy slender forms. The F strain infects mainþ

skeletal muscle, with a predominance of broad forms.

Bloodstream trypomastigotes

(BT)

werc

obtained from Swiss albino mice

at

the peak

of

parasitemia (?th dqy

for

the

Y

strain, and 35th day for the F

strain).

They'were separat ed from defibrinated blood by differential

cen-trifugation, washed, and suspended

in

M

199

(MedÍum 199, Flow Lab., U.K.), containing 20Vo

fetal calf serum,

at

the desired concentration,

as previousfy described 12.

Tissue culture trypomastigotes (TCT) were obtained from the supernatant of LLC-NIK,

mo-nolayers, infected

7

days previously, washeC and suspended as above.

Macrophages

_-

Peritoneal mairophages were obtained from Swiss mice on the 4th day

after

peptone stimulation, pooled, seeded on coverslips

in

Petri dishes and incubated f.or 24

to

48 hs

in

ML99,2OVo fetal calf

serum.

They

were then infected.

with

parasites

_{at a}

para-site-cell ratio

of

1:1. After 30 min

to

3 hs

con-tact they were ,washed _{and either fixed}

imme-diately

or

reincubated

for

an additional 48 hs

in

fresh medium,

all

procedures being carried

out as previousþ described r2.

Sensitization of parasites with mouse sera.

ï

strain TCT were suspended

in:

a)

normal mouse serum;

_b)

plasma obtained

at

the ?th d:Ly

cf

infection

with

Y

strain trypanosomes,

180

or

c)

hyperimmune mouse anti-T. cruzi serum

at subagglutinating dilution (1:50). At this

di-lution an anti-mouse immunoglobulin

fluorcs-cein isothyocyanide conjugated rabbit

immtt-noglobulin revealed â very slight fluorescence.

After incubation

for

t

h

at 37'C, they'were

di-Iuted

to

the desired concentration and

put

in

contact with macrophages at a 1:1 parasite cell

ratio. for 3 hs.

Microscopy

_-

For light microscopiy

cover-siips were stained

with

Giernsa. Percentage of parasitized cells and number

of

parasites per 100 ceils were scored

in

500 cells 3 hs and 48 hs after parasite contâct. Electron microscopy

was made with

Y

strain parasites and speci-nrens were processed at 30 min and 3 hs

post-infection. Cells were washed ,with Hank's so-lution, fixed

"in

situ"

with 2o/o (v,/v) glutaral-deþyde. After scraping the cells

with

a rubber policeman thqy were washed again, fixed

in

1% osmium, deþydrated and embedded in Araldite. Sections were examined either

in

a Philips 301 or Zeiss EM-g electron microscone.

RESULTS

Figure

I

shows quantitative differences between interiorizatíon of BT and TCT of both

the

Y

and F

strain.

As can be seen, parasites

originating from tissue culture ,were

interiori-zed

by

macrophages

at

a

much higher per-centage than those originating from the blood-stream of mice. Initial infection rate with TCT

of both strains was higher than one (Table I),

while usually only one parasites per cell was seen

in

infections with BT.

Differences

at

the initial phase

of

interac-tion between

Y

strain

BT

and TCT

with

ma-crophages were also noted at the ultrastructu.

ral

level.

After

30

min

TCT were frequently seen in clusters at the area of contact with ma-crophages (Fig. 2). At this area the macropha. ges qytoplasm ,wa"s _{almost devoid of organelles} except

for

smooth vacuoles,

with

expanding pseudopodia indicating

an

active process of

phagocytosis, although the rnacrophage already

harbored one

or

more parasites

within

large

vacuoles. With BT

at

the same parasite cell

ratio

(1:1) we were

not

able

to

observe the attachment phase.

After

3

hs interaction, BT were localizecl

KLoETZEL, J' K'; MILDER', R. v. & UMEZAWA, E. s, _- Trypmosoma crui interaction with macrophages: Differences

between tissue culture and bloodstream forms. Rev. Inst, Meal, úrop. São pauto p6:1?9.1g5, _19g4.

TABLE I

Interiorization and multiplication of bloodstream and tissue culture trypomastigotes in macrophages Parasites

Origin

Bloodstream Tissue cultute Bloodstream Tissue culture

0/o cells _No. parasites/ _infection

infected 100 cells rate

(A) (B) (B/A)

40

830

(9

Í.o

fL

o

É.

lo

()

=O

o

l¡J

t-

₍₎

40

l¡J

2so

L

o20

òe 30.3 9.0 34.0

Y

STRAIN

c.ï

56.0 9.0 68.0

Time of interaction

1.0

1.84

1.0 2.0

o/o _cells

infected (c) Ð.ó 40.8 0 40.0

Figure 1 and Tabie

I

also present resutts a.fter 48 hs interactÌon. The percentage of ma_ crophages Ínfected with

Y

strain BT and. TOT

was

not

altered significantþ when compared

with 3

hr

interaction. However, with F strain

parasites

there was

a

clearcut difference be¡ween BT and TCìT: while TCT developed. and

multiplied

within

macrophages

this

did. not

happen with

BT.

This can be visualized more

clearly

in

Taþle

I:

after 48 hs interaction wlth

F

strain, macrophages

initialþ

infected. with

BT no longer harl¡oured parasites whÍle TCT survived and multiplÍed. Both BT and TCT of the

Y

strain multiplied within the host celts.

Interaction for. 3 hs between macrophages

and

Y

strain TCT which had been previously

incubated with normal mouse serum, ?th day

infection plasma or hyperimmune specific anti_ serum, did not present any significant differen-ces

in

infections rate among these groups.

DISCUSSION

The fact

that

TCT in-fect macrophages îo

a much higher degree than BT may be due t;o

some features

of

parasites, membrane. Thus,

tqypsinization

_of

BT results

in

infection rate comparable

to

that no,w observefl

f6¡

fef

o,r+.

One

of

the possible _{differences would be the} presence

of

host derived elements on the

pa-rasite's membrane, such

as

immunoglobulins

that are found on circulating

T.

cruzi parasi_ tes 7,8. Receptors

for

the

Fc

portion

_of

_IgG

have been described for T. cruzi 13 _and this _may

be one of the mechanisms which might inhibit phagocytosis.

_In

_{our system we ûere unable}

to

detect ar¡y significant influence on macro-phage uptake

of

TCT pre-incubated

with

nor-mal mouse serum, ?th day infection plasma or hytr¡erimmune serum

at

sub-agglutinating con-centration.

F

STRAIN

No. pâmsites/ multiplication

100 ceUs rate

(D) (D/B) _x (A/c)

to

a

o

16.5 209.0 0 198.0

Fig. 1 - Infection of macrophages 3

exposure to T. cruzi bloodstteem forms culture _{forms t#l}

348

HOURS OF

INTERACTION

3.09

2.76

0

an

amorphous, somewhat granular substance was also seen enclosed in this vacuole together

with membrane debris and flagellar fragments (Fig.

_3).

On the other hand, at this period, TCT

were enclosed

in

a

more clear,

tight

vacuole,

KLOETZEL, J. K.; MILDER, R. V. & UMEZAWA, E. S. _- Trypanosoma crMi interaction with macrophages: Diflerences

between tissue culture ând bloodstream fo¡ms. Rev. Insú' Med. trop. São Paulo 26:Ug-185, 1984.

FÍg. _{2 - 4 -} Electron micrographs of

T. cruzi Y strain. 2. Tissue culture

try-pomastigotes. 30 min interaction. Pâra.

sites (P) are seen inside the macrophage

ând outside the cell attached to the

ex-panding pseudopodia. X 28,500; Bar ₌ 0,5 p

182

FÍ9. _{3 -} Bloodstream trypomastigotes. 3 hs

in-teraction. Trypanosome within a "Iarge" phago.

cytic vacuole which contains an amorphous

ma-terial. _{f -} flagellum; _{K -} kinetoplast. X 82,000;

KLoETZEL' _{between tissue} J' K'; _{culture and} MILDER', R" _bloodstream v' & uMEzAw.{, _{E. s. -} lrJæanosoma cruzl interaction with macrophages: Differences forms. Rev. rnsú. Med, trop. são pauto 26:1?9-185, 19g4.

Our electron microscopic studies

of

fhe

early post infection events indicated that TCT penetrated macrophages by phagocytosis, _{as is} ciescribed

for

the interiorization of BTe,1t,r2.

The presence

_of

clusters

_of

parasites con_ centrated

_at

the macrophage surface corrobo_

rated the high phagocytic rate

of

TCT ,which

was also expressed

by

the simuitaneous pt'e-sence of more than one parasite inside the host

cell.

This parasite concentration

in

certain areas of the macrophage membrane

suggested-the existence of a preferential _{sufface area for}

the

attachment

of

trypanosomes. Whether

this represents specific interaction sites at the

host

cell

and/or

at

the

parasite surface

re-mains unknown. We were

not

able

to

study

this

phase

with

BT, since

their

phagocytosis

is much lo,wer, thus rendering the opportunity

to

visualize

the

event more

difficult.

Other Authors e were aþle

to

see the attachment of

Fig. 4 - Tissue culture trypomastigote. _{J hs} in-teraction. Cross section of the parasite within a

"tight" vacuole. _{F -} flageUum; _{K -} kinetoplast; MN _- mâcrophage nucleus; _{N -} paÌasite nucleus.

Microtubules are seen nearl¡y (arrow). _X 29,000;

Bàr ₌ 0,5 u

BT, using

a

10:1 parasite-cell ratio, but, even

at this high concentration only single parasites r,'"'ere _attached to _{the cell surface.}

_In

_{any case,} attachment process

_of

TCT seemed

to

be si_ milar

to

that

of

epimastigote culture forms 17

as 'well as

for

the culture forms

of

Leishma-nia 3.

Also

the way the

parasite

_is

eventually lodged

in

the phagocytic vacuole ,was _variable depending on the origin of trypomastigotes

en-volved. 'While at 3 hs BT were enclosed

in

a

loose vacuole

filled

with

dense material, the

TCT was enclosed

in a

clear,

tight

vacuole.

The meaning

of

these morphological differ-ences is unknown.

T. cruzi strains differ in their þehaviour in many aspects, and several features, such as parasitemia, mortality

in

mice and the

KLOETZEL, J. K.; MILDER,, R. V. & UMEZAWA, E. S. _- Trypmosoma cruzi interaction with macrophages: Differences bet'ween tissue culture and bloodstreâm forms. Rev. Inst, Med. trop. São Paulo 26:179-185, 1984.

metimes ascribed

to

some morphological

cha-racteristics

of

these strains, i.e., the predomi-nance of broad or slender population2. The CL

strâin, with predominance

of

broad forms, is

reported to have low infectivity

for

macropha-ges, even rvhen TCT are emplqyed, suggesting

that this may be a characteristic of the strain 10.

The F str.ain employed in the present paper is

also

a

broad population

strain

and

BT

are

destroyed

in

macrophages

"in

vitro" l2.

How-ever, 'when comparing

BT

with

TCT

of

this

strain, we verified

that the

latter ,were able

to

multiply within

macrophages,

and

there-fore their

behaviour

is

distinct

from

CL

pa-rasites.

'We may conclude

that the

capacity

to

multipfy

or

not

within macrophages may

be

inherent

to

the

trypanosoma strain itself,

but depends also on the parasite's origin; thus,

rvhile'with Y strain, both BT and TCT multiply

in

macrophages, with

F

strain BT are rapidly

cÍestroiyed

by

macrophages and only TCT are

capable

of

multiplication

within

these cells. RESUMO

Interação ehtre Trypanosoma cruzi e

macrófa-gos: diferenças entre tripomastigoúas sangüí-colas e de cultivo de tecidos

Macrófagos obtidos

do

peritoneo

de

ca-mundongos após estímulo, com peptona,

fo-ram cultivados em lamínulas, infectados com

tripomastigotas das cepas

F

e

Y

de

T.

cruzi,

obtidos de cultivo de tecidos ou do sangue t).e

camundongos infectados. Os parasitas,

obti-dos de cultivo de tecidos, tanto da cepa

Y

co-mo os da cepa

F,

são interiorizados

por

ma-crófagos em proporção muirlo mais elevada do

que os sanguícolas. Parasitas

de

cultivo de

tecidos incubados com soro de camundongos

normais, ou soro hiperimune específico em di-h.rição sub-aglutinante, comportam-se

essencial-mente como parasitas náo opsonizados. :Fo-ram observadas diferenças

a

nível

ultraestru-tural na fase inicia,l de interaçã,o entre macró-fagos e tripomastigotas das duas origens. Após 30 minutos, tripomastigotas de cultivo de

teci-dos localizam-se em agrupamentos na área de

contato com os macrófagos. Enquanto os

tri-pomastigotas sanguícolas estáo na maioria das vezes

no

interior de vacúolos fagocíticos lar-gos, após 3 horas de interação os

tripomasti-gotas de cultura situam-se em

um

único

va-cúolo estreito. Tanto as formas de cultivo de

184

tecidos quanto os tripomastigotas sanguícolas

da cepa

Y

multiplicam-se ern macrófagos; os tripomastigotas sanguícolas da cepa F sáo

des-truídos

no

interior da célula hospedeira,

en-quanto os tripomastigotas de cultivo de teci-dos desta cepa são capazes de multiplicar-se.

REFERENCES

1. ALCANTAR'A, A. & BRENER,, Z. _- Trypanosoma cruzi: role of macrophage membÌane components in the

pha-gocytosis of þloodstream forms. Exp. Parasit. 50:

1-6, 1980.

2. BRENER,, _{Z. -} Immunity to Trypanosoma cruzi.

Advanc. Parasit. l8: 24?-292, 1980.

3. CHANG, K. P. _- Leishmania donov¿ni: promastigote-macrophage sudace interaction in vitto. Exp, parasit. 48: 1?5-189. 19?9.

4. DEANE, M. p. & KLOETZEL, J. _ Lack of pro-tection âgainst Trypanosoma cruzi by multiple qoses

of T. lewisi culture forms. A discussion on some

strains of "lewisi,,. Exp. paræit. 85: 406-410, 19?4

5. DVORAK, J. A. & SCHMUNIS, c. A. _- Trypanosoma

cruzi: interaction with mouse peritoneal macrophages.

Exp. Paræiú. 23: 289-800. 19?2.

6. KIPNIS, T. L.; DAVID, J. R.; AIPER,, C. À.; SHER,,

A. & DIAS D.A, SILVA, _{W. -} Enzymatic treatment

transforms ttypomastigotes of Tr¡/panosoma cruzi into activators of alternative complement pathway and

po-tentiates their uptake by macrophages. proc. Nafl. Acad. Sc, USA ?8:602.605,1991.

?. KLOETZEL, J. & DEANE, _{M. p. -} presence _of im_

munoglobulins on the surface of bloodstream Trypa.

nosoma cruzi. Capplng during differentiation in

cul-ture. Rev. Inst. Med. trop. São paulo lg: Bg,t_402, tg77.

8. KR,ETTLI, A. U. & NUSSENZWEIG, R,. S. _ PIE.

sence of immunoglobulin _{on the surfâce of circula[mg} try?omastigotes of T. cruzi resulting in activation of the alternative pathway of complement and 1ysis. !Vas_

hington, P.A.H.O. Sci. publ. g4lt ,tI-78, _tg,tl. 9. MAR,IA, T. A.; ALCANTAR,A, A. & BR.ENER, _z.

-Ultrastructural studies on the in vitto interaction of

Trypanosoma cruzi bloodstreâm forms and mouse

peritoneal macrophages. Acta trop. 89: 99-109, 1992.

10. MEIRELLES, M. N. L.; CHIAÌ,r, E. & SOUZA, W. _ fnteraction of bloodstream, tissue culture-derived and

axenic-culture-derived trypomastigotes of Trypanosomâ

cruzi with macrophages. Acta frop, 39: 19b-203, 19g2.

11. MILDER,, R. & KLOETZEL, _{J. -} The development

of Trypanosoma cruzi in macrophages in vitro. Inte-raction with lysosomes and host cell fate. parasito_

logy 80: 139-145, 1980.

12. MILDER, R,.; KLOETZEL, J. & DEANE, _{M. -}

KLOETZEL' J' K; MILDER', R. v. & uMEzarffA, E. s. _- Trypmosoma cruz¡ interaction with macrophages: Differences

between tissue culture and bloodstream forms. Rev. Inst. Med. trop. são paulo 26:1?9-185, 1984.

witlì Trypanosoma cruzi. U. Intracellular fate of

bloodstream forms. Rev. Inst. Med. úrop. São paulo 19: 313-322, 1977.

MInANDA-SANTOS, I. K. Fer¡eira de & CAMPOS

NETO, _{A. -} Receptors for immunoglobulin Fc on

pathogenic but not on nonpathogenic protozoa _{of the}

Trypanosomatidae. J. Exp. Med. 154: lIB2-t742, L1BI.

NOGUEIRA, N.; CHAPLAN, S. & COHN, _{Z. -}

Try-panosoma _cruzi. Factors modifïing ingestion and fate

of blood form trypomastigotes. J. Exp. Med. 152:

44?.451, 1980.

L4

15. SANDERSON, C. J. & SOUZA, W. de _{- A} morphoto. gical study of the interaction between Trypanosoma

cluzi and eosinophils, neutrophils and macrophages

in viúro. J. Cell Sci. 37: 2,t5-286, 1979.

SILV.A, L. H. P. da & NUSSENZWEIG, _{v. -} Sôbre

uma cêpa de T. cruzi altamente virulenta para o

camundongo branco. Folia Clin. et BioI. (S. pauto) 20: 191-208, 1953.

TANOWITZ, H.; WITTN,ER, M.; KRESS, y. & BLOOM,

B. _- Studies of the in vitro infection by Trypanosoma

ctuz¡, I. Ultrastructural studies on the invasion of macrophages and L-cells. Amer. J. trop. Med. Hyg. Z,4t 25-33, L975.