T-lymphocytes recognise Leishmania glycoconjugates

P;lraato/ogy 7odq voi 1,no 2. i 985

61

T-lymphocytes recognise Leishmania glycocon jugates G.F. Mitchell and E. Handman J-lymphocytes can have erther resistance-

glycocon+rgate (L-GC).

promotrng or &ease-promoting

lrprd and orrented

effects ,n

murrne cutaneous letshmanr~s~s.Recently we

that

showed

o /rp&contor?lng

giycocoqu-

MHC

molecules

rophages,

when anchored by

In relation on

We

to class I/

infected

mat-

also propose

sron of T,,

cells by consumrng lnterleukrn-

2 (IL-2). These ‘suppressor’ T-cells have a

IS both an Inducer and a target

of

promastigotes to mocrophages. wrll vaccinate mice against disease. in contrast,

T-cells. These cells lack the Ly2 surface antigen and can actrvate macrophages;

trons from

the

they must be present at hrgh frequency

mrce”’ ‘j. These

water-so/jib/e

carbohpdrale

fragment

certain

class

It

normal

[or suppress/on ofhost

T-cells can efficrently restore

resrsfanre We prop-

ose that two types ofi-cells suface

anflgen,

eff+cts in cutaneous drferent

lackrng the Ly2

and with fwo very d@rent iefshmanrasfs, recognize

carbohydrate

epr[o’>es on the same

i

<.T,,, are

readily

molecule IS orrentated

presented to the rmmtlne system

the ltprd component rophage

Isolates of the p-otozoan

para-

nude

membrane

leased from T,,

when

The

suppressor

carbohydrate

process,

infected mac-

anchored

Into the promastrgote

molecule

mernbrane by covalently bound llprd and IS the r-ecognltron molecule for macro-

rem

cells which activates the

phagesl’

Further-

the glycocon+rgate

promoting

sponses and the generation

Irpldated glycoconlugate

may be y-Interferon,

actrng

of parasrto-

Tcells

Such mace show striking varratrons In sus

crdal oxygen IntermedIates In Infected mat-

receptors

ceptrbrl~ty. In BALE&

rophages’.

which are recruited

strains (H-2

mrre

and

other

congenrc strarns), expanding

If the expression

of class II MHC

or avatlabrlrty

chronrcally

rnjectron

genetrcally susceptrble BALBIc mrcel.1, then

sequent

of

vrscerallzatlon

death Other are

promastrgotes.

with

of infectron,

suband

mouse strarns. such as CBA/H.

hrghly resrstant to

small cutaneous

disease

Infected

the efficiency of MHC-restrIcted sron of T,,

uninfected

de-

on the

macrophages,

Into the lesion by l--

rty reactrons ’ The

expres-

Ly2_ rewance-promoring

L-GC

at the surface of promasr-

gotes, and perhaps

at the Infected

mat-

rophage surface, IS susceptrble to cleavage

Ly2- disease-promoung

Tna cell at high frequency.

completely’

spec,fic,ry for recognltlon-

I, characterrstcs

with

(IXGC)

and any

lessons or swellings that

host

of

cells IS r-educed I’) I’.

appear at the tlllectron sate heal rapidly and Two

macrophages

can occur

cells which mediate delayed hypersensltrv-

molecules IS drmrnshed on

cutaneous lessons develop after cutaneous

of

IS

surface

rnductron of disease-

macrophages,

can infect mace of vartous Inbred strarns.

In this at-

tachment

at close range and leading to oxrdatrve re-

lershmanrasrs,

of

of

trc (Old

cutaneous

determinants

mastrgotes to gain access to mat rophages

sate, Lershmanro major, the cause of zoonoWorld)

BAL&

T-cells also

through facrlltated phagocytosr9

the

by IncorporatIon

Intothe

rn cell pop&

Infected

and bound to a macr-ophage receptor [Frg I ) This treceptor IS used by L. map pro-

portton of the L-GC avarlable

molecule depending on how thusn-to/ecu/e IS

Human

recogntse

chronrcally

the glycoconJugate, tn this case de-llpldated

cells recognize determinants

on the carbohydrate that

rn

resrstance to

In hypothymrc

In normal lymphold organs

but are at a high frequency

lymphord organs because normal

mc?ot rnfectlon

mlceg

low frequency

MHC-restricted

denved from thn glycoirprd w~/lsensltrze mice

drsease-

T-cells” lo Id I0 tnhrbrt expan-

gate, involved in
that Ly2-

promoting

affect

course of rnfec?ton: T-cell-dependent

the

T

cell at low frequency. _

spec,fic,ty for backbone

ste ep\topes of

epnopes of glycocon)ugare EZ

glycocon~ugate

(htgh clonal exparwon

rm-

MO

A--\

IL-2 connumlng potentlall

mune responses, and the ‘permrssrveness’ of host macrophages3.

As expected,

the

characterrstrcs Iof the parasrte also Influence the outcome

elf rnfectlon”m’, and there are

cloned lines of i. major which do not produce

lessons In genetically

susceptjble

mice> a. Although sial, the

several Issues are controver-

cellular

cutaneous

rmmunolcgy

lelshmanrasrs

clearer. There lymphocytes

of murlne becomrng

IS

IS no doubt

that certain T-

orientedby

pr-omote both the perpetua-

Macmphage

lipid.

tion and the resolutron ofdrsease9 ‘0. In this artrcle, we argue that an amphrpathrc, Irprdcontarnrng glycoconfugate the de-lrprdated,

of L. ma/or. and

water-soluble

glycocon-

fugate derrved from It by enzymatic cleavagel’ 12, have cutaneous

a central

role

lershmanrasls. We

each stimulates a drfferent

In murrne belreve that

subset of T-

Fig. I _Pi oposed scheme of events resuhng inInductronand exprcssron of resistance-pr-ornot~ngand disease-promoting rmmune responses in murlnc cutaneous lershmanlasfsE r-eprcsents class II MHC-rrestrrcted rnacrophage actuation It close range. which may be rttlated by y-interferon. II MHC

Ei represents

(I rqon)

restricted

macrophage in both

~nh~b~t~onthroqh

IL-2

consumpt:on

lnducnon

1s class

casts

cells, one responsrble for the Inductron and expression and the promotrng We For

of host-protec:rve other

responsrble

rmmunrty for

drsease-

immunrty

propose

Abbreviations thymus-der excreted

I, InductIon lved cell, MHC.

E.

expression,

malor

i,,,

cell,

macrophage-actlvdtlng

h~stocompat~b~l~ty complex

[H-Z).

l-cell,

T-cell,

11-2, ~nterlcuk~n-2.

EF,

factor

that the Irprd-contarnrng

technical reasons we

are unable

toreproduce this figure in colour in this edition -

see the August

issue

of ParasitologyToday for full dour

illustration

62

Pswtoiogy

by an enzyme wrth a phospholrpase CIIIllke activrty In L. ma/or’ ‘,‘2. The carbohydrate-nch DL-GC, together with any LGC In promastlgote culture supernatants are what have been termed ‘excreted factor’ (EF)22*3. These molecules form the basrs of’s serotyprng system In Lelshmonia spp. first described by Schnur et 0f.2~.Parasites with high enzyme actrvrty WIII cleave L-GC raprdly and, as killed organisms, ~111 vaconate less effectrvely than parasrtes with low enzyme actlvlty2s. EF has Inhrbltory effects on responses of Immune human blood lymphocytes to crude promastlgote antrgens, responses that surprisrngly can be ascnbed entirely to periodate-sensltlve determlnantslb. As dlscussed prevlously2. the range of specrficltles that disease-promoting T-cells may have In this system IStotally unknown. We have emphasrsed rn thrs hypothesis a population of Ly2-- T-cells at low frequency In normal uninfected mice that has specrficrty for carbohydrate backbone eprtopes of DL-GC. We propose that these T-cells expand rapidly and are potent IL-2 consumers. This IS speculatrve, but could explarn the activity of Ly2Y cells that lnhrblt clonal expansron of T,, cells which lack Ly2 markers2. If this notlon IS valid then It may be that any T-cell populatlon with high IL-2 consuming potential and which IS specific for any antigen determrn ant, may have disease-promoting potentral. The speculation could further suggest that Ly2- T-cells at low lnrtlal frequency In normal lymphold organs, as dlstlnct from those at high frequency that have already been clonally expanded through Internal lmmunoregulatory clrcurts will have many IL-2 receptors. That IS,there IS an Inverse correlation

between

the srze of a ‘resting’

T-cell clone and the numbers ceptors

high frequency, further

of IL-2 re-

on each cell. TMA cells already at yet requrred to proliferate

In order

rophage

to mediate efficient mac-

actlvatron

(at

mice). are dependent

least

In BALB’c

on high IL-2 levels

number the

of MHC

surface

molecules IS reduced on

of Infected

BALBic mlcei4. This presumably applres to fected, macrophages and, In terms of Ly2T-cell recognrtion. to class II MHC molecules. molecules IS nude mace Infected with low numbers of normal cells where the actlvltres of the T,, cellsare unln terrupted by disease-promoting T-cells The latter are present In very low numbers In mlnlmally reconstituted nude mace. Thus effect of parasl%atlon In BALE’c mice becomes increasingly Important as the efficlency of T,, cell inductron and expansion IS

BALlVc

and other

ible mice might be responders the

macrophage

whereas

on

most other

are genetically

receptor mouse

(Fig.

I)

strains that

resistant. such as CBA/H.

are nonresponders.

lmmunrzatlon

with a

view to ellcrtrng ant/body to DL-GC

(or to

basis of

In CBA/H.

non-responsiveness

may hinge on the per-

mrssrveness of macrophages’

mice and close relatives compared mace such as CBM,

C3HHe

WI&

and C57BL/6?

I+, the para-

macrophages

medrators30”. ferent

to

patterns In the Inheritance

of resis-

tance In F, hybrrds of BALBic and various resistant parental stralns’3. Analysis of the I-GC GC

and derived

DL-

has been facrlltated by the avallablllty

of two monoclonal

dntrbodres (Mabs) di-

One

determinants

techniques

Mab, WIC79.3 We

and

using Mabs’ I.

has been used exhave

found

that

Fab

the lnfectrvrty of promastlgotesa4.

radrormmunoassays

to demonstrate

In the sera of vaccinated, ceptrble

the

of antrglycoconfugate antrbodre; mice

that are

easej5 36. AntIbodIes

genetically susresIstant

to drs-

are not detectable

sera frorn chronIcally Infected

In

mace. The

Any explanatron must take account of the

hrgh levels of antrbody nated

BAL’Bc nude mace promote

actrvrty to L-GC with some helper actlvlty

mrnrmally reconstituted

BALB’c nude’), and

the protective of normal

BALBic mice

or EF WI/I reduce

effects of small numbers

BALFYc cells In nude mace. To

date, thrs IS the only evidence that dlseasepromoting

T-cells in this system may In-

clude cells with speclficlty for DL-GC are

directed

to

carbohydrate

whrch doffer from I-GC tion to the Immune must be protected

in therr presenta-

from enzymatic degr-a-

1%vacclnatlng

that IS protective

L-GC

that

eprtopes

system. Thus, L-GC

datlon In order to preserve

in the

in vaccl-

nated mice IS still IndIrect and WI/I remain can be synthesized.

the avaIlable data entourage

based

against on the

cutaneous

lelshman~asrs

lipId-contalnrng

glycocon-

jugate of i. major Acknowledgements:

T-cell-derived

This could lead to quote dif-

fact that small numbers of normal T-cells In resistance -the

with DL-GC

sate Isolate used, and responsrveness of In fected

presence

more eficlent In BALBic

Interest IS the recent de-

that cells from

vaccine

of this complexity

reduce

IS the process of T-cell-dependent

monstration

of EF and

major antlbodles38.

the vigorous pursurt of a defined antigen

Some

The Mabs have been used In competrtrve

Why

polyclonal rabbit an&i

Injected with L major IS complexI..

--$ hrgh resting clone

disease promotlon

will antigen antlbody complexes

rbrllty versus resrstance In varrous F, mice

low--j

high

EF cross-llnked

WIII protect C3H mace agarnst dlseasejj as

Nevertheless

of sustept-

bated with low numbers of promastrgotes

with low +

However,

with muramyl dlpeptrde and poly-L-lyslne

though the Issue of dominance

are

low IL-2 pr-oductron.

not pro-

lelshmanrasls by

Injections of isolated llpld nor of EF, contarnrng DL-GC.

so until the molecule

of IL-2. The range of

size would then equate

against cutaneous

Such FI mace are resrstant to L major al-

both low producers

+

or Freund’s

Mice are

purified

was the

fragments of thus IgG, Mab when prerncu-

rntermedtate

tected

adluvant’4.

tant 11‘self tolerance’29 to DL-GC

and

frequency)

complete

efficacy Evidence that tt IS the L-GC

if both

T-cells (low

mace. against disease

when Injected wrth C pan/urn

EF) has always been considered very drfficultjs. FI progeny from BALM x CBA/H mice would be suscept\ble If an Immune response gene for DC-GC reactlvlty Inherited from the BALBc parent was the basis of responsiveness, or they would be rests-

tenslvely32 I3

ant&DL-GC

congenlc mace, and genetrcally C3HMe

Immunized

to DL-GC

on Mab columns

promastrgotes

Of particular

genetlcally suscept-

Ly2-

reactive T-

will vaccinate genetlcally susceptible BALI% and

suming T-cells of the other type

easrer to envrsage thts sequence of events (high frequency)

affinity-punfied

solublllred

BALBlcH-2

lmmunoblottlng

anti-L-GC

L-GC from

resrstant

reduced by clonally expanding and IL-2-con-

disease-promoting

I985

cells

of no consequence In BAL&

allable, T,_,, cells may become

It 15

of suppressive,

T-cells and lnhlbrtron of L-GC

This decrease In surface MHC

rected to carbohydrate

to specrfic unreactlvity or tolerance2’.

of

chronlcally Infected, rather than recently In-

because they bind IL-2 poorly. If little IS avvulnerable

macrophages

1,no 2,

Today, voi

In resistant, vaccl-

mice may reflect high Ly2-

for antibody productIon.

the sublethally lrradrated BALBic’“, are rests-

of antrbody

tant to chronic cutaneous lelshman~asls.The

fected

T-cell

In contrast, a lack

In sera from

chronrcally In

mace reflects the domrnant actrvrty

8tudles it-om th,s labors

atory are supported by the Australw Ndtlonal Health and MedIcal Research Council, the UNDPZr\/orld BankWHO Spec~dl Programme. the Rockefeller- Foundation, dnd grant No Al- I9347 from the National Institutes of Health, U 5 A

P;iras~toiogv Today, voi 1. no 2. 1985

9

63

Al;st / txp BIO/

MNtchell. G F et ai I I98 II

Mcd So SO,539-544 IO

Clew

I Y, I-ialc C and Howad

lmmu~~ol II

I 28. I9 I7

Handmdn,

JW I2

t

Ii

Mitchell.

I4

Handmar

: 1979) I5

t

22

Zehavl.

Goding,

imrntnogenet

Crrcdlg.

EURO

immLro/

JA

K

and

! 0, 395 G F

21

Cd

25

MItchelI.

( 1982)

8

C B

and

133, 335 I

1

Schrur

; l9?1)

Wyler

D]

26

Zucker “UT

G F

I9

TltLs

JC,,Hale.

Revi 6 I 2 Ii-

C and xr*r.

eta/

(l984)/

(i 98 I )I

FY

irwnunoi

Pzasitenkd 69 695-

A and Gr eenblatt

C L

Handman

t

27

Malkovsky

M

28

133, l594-

Vllth

29

and Medawdr

31

Nacy.

J

(I 979)

EXQ

Schnur.

LF

32

and

C1983)

CL

a/

~1983)

Meltzer.

l:nmiinof

&ii

immun

77,

lbarra,

A AL

Pormrtology

de, Howard

!otiler.

AH

J G and Snap-y. D

85 523-53

C i

M,crobrol 34

M 8 and

133. 3344- 3350

i 1982)

Greenblat

33

et

I

a;

J

Cifn

i 1985)

Proc

t 1983)

18, i91 -191

Handman,

E and

Not1 Acod

Sci USA (in press)

MItchelI

35

( 1984)

Mitchell.

Greenblatt

C

txp

I

Ecdysone

Handman.

G t

G F

E and ipithill.

BIOI Med

( 1983)

I 53

4115r ]

I, I l-25

So 6 A

E

T W

!-li-

Bn! Me0 iit 62

and Handman

Steinbet-ger

pr (11 (I 984) E$

Poro~~tol 58,

223- 229 38

ASM Nee

G F

A,,sr / &I

MItchelI,

36

37

Porosirot47, 254-169

Grrenblatt

a

CA,

: l984)/

P B (I 984) Inl~

Today 5, 34G342

El-On

CA

298- 307

and Spzthill, T W

249-256

Exp Med 153 ii-?-568 KG

14)

p V c! toi

wnology

Ho:vatd

(Suppl

Ponnroi (III press) ( I9831 Porl~~rte frwwnol 5

Inf 1

Londner

243 I8

Qtof

Med 5~s 8 ?iz? 941

isr /

MItchelI

3357

, l981)lmmonoi

rtdi

Rev

etcn(I 98i),r

U

\ 1985)

Panoi~an.

;I 983) lot

493 Nacy,

30

632

Her nander A G ( 1983) ,n CkTopdtholo#in hrasitic niseasrs CIKA ~o”n~iarlorlSyw poi~um99 PItman, London, pp 138-l 56

23

6~’ 74 82

98-l)/

KP

305 630

701

2306

j W ! 1985)

R M and MxF:ae.

JP

Lou!~,

ano

Aust / ixp 510’ Mtd SC, 57, 9-29

Sypek

I7

Chang,

i,2301

f cl983)j

G

lrnmi~r~ol

,

21

1.29 336

Cot-cry&

I6

JG (1982)j

(I 983) niarure

267-305 C L

I! and God,ng,

/owrl‘?/ 4

L~ew i-Y

I922

GI-eenblatt

(1984)fMHO/ournal

Handman

I600 20

Handman.

49, 48%

i

e: (11 (I 477)/

Przorool

(SuppI)

24,2lA

workshop

J.G. Mercer Edinburgh:

3 I st March -

3rd April

1985.

be important If circulating ecdysterolds are

ety of arthropod

to form the basis of dlagnosls.

P. DIehI; T. Crosby etal; K. Hoffmann. Unl-

Variation during

Although

ecdysterolds

(rnoultlng

hor-

In ecdysterold

cestode

examined

In

concentration

development the

rat

was

versltit

Ulm,

Unlverslty

tapeworm,

species (1-L. Connat and

FRG; A. Slinger and E. Isaac,

of Leeds).

The regulation of ecdysone synthesis by

mones) were orlglnally characterized In ar-

Hymenoiepis dlmlnuta (J. Mercer et a/.. Uni-

prothoraclcotroplc

thropods,

verslty of LIverpool).

the tobacco hornworm,

they have now also been de-

Three

developmen-

tected In coelenterates, anrellds, molluscs,

tal stages of the parasite wIthIn the define-

provoked

trematodes’,

tlve host were analysed and I O-fold varla-

that

tlons

prothoraclc

and

cestodes2, and In free-llvlng

parasttrc

While

nematodes’

the

In free

ecdysterold

physIologIcal function of h<:lmlnth ecdys-

wer-e recorded.

terolds

ences In ecdysterold

IS unknown, their detectton In the

concentration

Although

these

dysone Workshop

consider ed the blosyn-

thesis, dlstrlbutlon

and metabolism

of ec-

dysterolds,

lnteractlcn

other

their

hormones

with

and their mode of ad;ion at the

level of gene Inductlo&epr?sslon. J. Koolman, lnstltut der Pillllpps-Unlvers&t, of

employing

an ecdysterold

radlolm-

in VIVO.

demonstration

using

radioactIve cholesterol. The

first

of

PTH

to

cyclic nucleotlde then stlmulattng ecdysone synthesis (W. Smith and L. Gilbert slty of N. Carolina).

Intermediate

thesis of ecdysone from

In the blosyncholesterol

was

Ecdysone

was also stimulated

Unlversynthesis

in vrtro by a 30 kDa

protein from larval haemolymph (D. Wat-

def?hrtlvely IdentIced as 7-dehydrocholes-

son and W. Bollenbacher, Unlverslty

terol by N. Mllner and H. Rees, Unlverslty

Carolina),

of

Marburg FRG, dIscussed the efficacy

direct

appllcatlon

glands sttmulated an Increase

In Intracellular calcium concentration which

suggestive of ecdysone biosynthesis awaits

much Interest. It was proposed

the

In turn enhanced cAMP synthesis, with the

this

Ec-

LIverpool.

while

radioactive

In

dlffer-

non-specific

Vllth

(PTTH)

Manduca sexta,

concentration were

body fluids of Infected hosts may facllttate diagnosis’ 3, --he

hormone

tncubatlon

precursor,

(2,22,25-tndeoxyecdysone),

of

the

possibly

Inter-endocrine

of N.

part of the complex

regulation

5-p-ketodlol

and juvenile hormone

with various

lenbacher et al.).

of

synthesis

ecdysone (W

Bol-

munoassay In the diagnosis of human hel-

embryonic

month tnfectlons, with analys s of sera from

mrgratorra did give rtse to ecdysone (M.

the meeting WI/I be published In a special

patients with c.llnlcally diagnosed schlsto-

Melster et ai, University

Issue of insect B/ochematry

somiasis,

bourg).

echlnococcosls,

hjmenoleplasls.

ancylostomlasls,

ascarlasls, strongyloldlasls

or

lmmunoreatilve

trlchurlasls

material

and larval tissues

L. Pasteur, Stras-

In addition to free highly

polar

of Locusta

ecdysterolds

conlugated

and

ecdysterolds,

was detected at different concentrations In

which themselves

the sera of 85%

for a long period due to their very low Im-’

comparatively

of Infected patients, and at low concentrations

of ‘uninfected’ controls.

The

in 5%

concentra-

tions of circulating Immunoreactive real suggested

rapid excretion

mate-

of ecdys-

munoreactrvity, dysterold

remained

undetected

metabolltes

Identified. These

compounds

were orlgl-

as ecdysone

roxyecdysone

estenfied at Czr with palm\-

ecdysone

Intravenously

mice (R. Lafont et a/., CNFS. proximately excreted

60%

Into

Paris). Ap-

of the radIoactIvIty was

In faeces or urine wIthin

mostly

as unmetabolized

though

metabolltes

less

48h,

ecdysone,

al-

polar than ec-

dysone were also detected This effclent excretion and metabolism WIII obviously

Nlrde.

or

P et al

(I 984)

\n Elosynthesis,

M&b&m

andModeof Actionof InvertebrateHormones (Hoffman.J and Porchet. M edsj pp 33 I 337, Sprqer-V&g.

nally detected In tlcks4b, and have been characterized

radIoactIve

References

has recently been

terolds by the parasites. The fate of ecdysby qectlng

Julran Mercer is at the Department of B/ochem/stry, Unwers~ of iwerpooi, PO Box 147, iwerpoolL69 35X, UK

a new class of apolar ec-

terolds

in VIVOwas examined

Some (but not all) of the papers from

Heldelber

g

Rees, H H. and Mendls, A H W (I 984) ,n Biosyn-

20.hyd-

Met&&m dndModeofActionofinver-

theas.

t&rateHormones (Hoffman,

J and Porchet,

eds) pp 33%345.

and P. DIehI, CNRS,

Biosynthesis.Metabolism andModeofActionof invertebrateHormones (Hoffman, J and Por-

Unlverslty

Koolman.

Pans; T. Crosby et al.,

of Llverpool).

On

hydrolysis

with pig liver esterase, these compounds yield free ecdysterolds to

be

either

metabolltes.

storage

or

Connat.

InactIvatIon

Slmllar, If not Identical, corn-’

pounds have now been detected In a varl-

J Walter,

chet. M. Heidelberg

and are presumed

Gen 5

eds) J -L

Camp.

W~gglesworth,

(I 985)

Springer-V&g,

M

tic, steanc, olelc or llnoleic acld(l-L. Connat

Arch

pp

Dlehl,

]

Heidelberg

and Zahner,

323-330, PA

H

( 1984)

r

Sprtngerer-Verlag,

and MO~ICI. M

(I 984)

Endocrinoi56, I oo- I IO K P, Lewis, Insect

D

and

Bnchem Physiol

Rees, H H I, 39-54