Nov 14, 1985 - A., Burmester,. OR.,. Waters,. S.J., and Winchester, ... Cell Immunol. 89,1, 1984. 17. Moss, J.,Fishman, P.H., Manganiello, V.C.,. Vaughan,.
Journal
of Leukocyte
Biology
40:65-72(1986)
Ganglioside Identification on Human Monocyte Membrane With Clostridium per fringens Delta-Toxin J-M.
Cavalllon,
C. Jolivet-Reynaud,
Unite d’lmmuno-Allergie (UA 557 CNRS) (C.J-R.,
C. Fitting,
B. David,
(J-M.C., C.F., B.D.), and Unite J.E.A.), Institut Pasteur, Paris
and
J.E.
des Antigenes
Abut
Bact#{233}riens
Clostridium
perfringens delta-toxin was first described as a hemolysin with a restricted lytic spectrum. A selective cytotoxicity of the delta-toxin was then found on rabbit leukocytes: peritoneal and alveolar macrophages were uniformly killed, whereas thymocytes were essentially resistant. The toxin was shown to be specific for GM2 ganglioside or a GM2-like structure. In the present study we report the interaction of delta-toxin with human monocytes. A specific, saturable, and irreversible binding of 1251-delta-toxin was demonstrated. Binding was inhibited by preincubation of the radiolabeled toxin with GM2 and with high amount of GM1 ganglioside. As judged by dye exclusion, no cytotoxicity was observed on freshly isolated monocytes, but when added at the beginning of a culture of human adherent cells, the cytotoxic effect was detected after 48 hours of culture. Taken together, these data indicate the presence of monosialoganglioside(s) at the surface of human monocytes, and suggest a possible reorganisation of such structure into the cell membrane when monocytes mature in vitro toward macrophage-like cells. Key words:
macrophage,
binding,
human
INTRODUCTION It has been recently shown that the binding of Clostridium perfringens deltatoxin to sheep erythrocytes [9,11] or rabbit macrophages [10] was the primary event leading to cytolysis by the toxin. Binding inhibition studies with gangliosides showed that GM2 gangliosides or a GM2-like structure was involved in the interaction of deltatoxin
with
cell
membrane.
Gangliosides, teristic constituents Received
November
a group of sialic acid of plasma membrane. 14, 1985;
Reprint requests: J.-M. 75724 Paris Cedex 15.
©1986
Alan R. Liss,
accepted
Cavaillon,
Inc.
February
Unite
containing Membrane 26,
glycosphingolipids, gangliosides may
are characact as recep-
1986.
d’Immuno-Allergie,
Institut
Pasteur,
28 rue
du Dr.
Roux,
66 tors
Cavaillon for
interferon
Mainly
cells
present
[3], in the
of the immune
or macrophages
and macrophages in some
system
immunologic can be delta-toxin
structure
at the surface
side composition
MATERIALS Delta
of the cells.
is poorly
AND
and
shed by certain might be used
monocytes
virus
[4], or bacterial
gangliosides
such as thymocytes
phenomena
that
to human
system,
[15]. Surface receptors [21] and are probably
gliosides perfringens toxin
[5,18],
hormones nervous
et al
are
also
toxins
[8], lymphocytes
for
gangliosides
involved in the
In this respect,
occur
of
NK cells
effect
of free
gan-
binding of Clostridium the presence of a GM2
we investigated in vitro
surface
[28], on lymphocytes [25] interaction observed
immunosuppressive
and macrophage-like
at the
[24],
in the cellular
cells [131. Thus the as a probe for assessing
[10,12,17,20].
found
derived
the binding cells
whose
of the ganglio-
known.
METHODS
Toxin
Clostridium perfringens delta-toxin was purified as described previously [2]. Preparation of ‘25I-labeled delta-toxin was performed according to the chloramine-T method [11] with ‘25INa (16.85 mCi/sg of I; Amersham-UK). Adherent
Mononuclear
Cells
From
Peripheral
Blood
Venous sion Sanguine, anticoagulant.
blood was collected from healthy donors (Centre Paris) in glass flasks containing acid-citrate-dextrose The blood was centrifuged, and the platelet-rich The cell pellet was diluted 1:1 in 199-Medium (Institut Pasteur layered on lymphoprep (Nyegaard & Co., Oslo, Norway). After 20 mm, 22#{176}C),the peripheral blood mononuclear cells (PBMC) monocytes
were
identified
by nonspecific
esterase
(NSE)
National de Transfuplus heparin as plasma was removed. Production, Paris) and centrifugation (400g, were recovered, and
staining.
PMBC
were
plated
cells per well in Bio-Products, Walkersvile, MD) supplemented with antibiotics (streptomycin 100 ag/ml; penicillin 100 lU/mi) and 10% fetal calf serum (FCS) (Boehringer, Germany), then incubated for 1 hr at 37#{176}C.Nonadherent cells were removed by vigorous washing.
in tissue culture (Nunclon 0.5 ml RPMI-1640 (M.A.
Binding
Assays
and
delta,
Competition
24 wells,
Denmark)
at 106 NSE
Experiments
Assays were performed as previously described [7,10]. Briefly, plated monowere incubated for 1 hr at 22#{176}C with either ‘251-delta-toxin alone, or with 125j.. delta-toxin plus an excess of unlabeled delta-toxin added 10 mm before the addition of the radioactive toxin. The final volume was 0.3 ml in RPMI-1640. The residual binding measured in the presence of an excess of nonradioactive toxin was taken to represent nonspecific binding. Specific binding of delta-toxin was defined as the difference between total and nonspecific binding. In the inhibition experiments that used gangliosides (GMI from Seromed, Berlin; GM2 and GM3 were a generous gift of Dr. B. Hauttecoeur, Biochimie des Antig#{232}nes, Inst Pasteur, Paris), ‘251-delta-toxin was added after a preincubation at 37#{176}C for 15 mm with the gangliosides. After washing, which removed the unbound material, the monocytes content (NSE cells) cytes
of adherent cells reached 90-95%. The ‘251-delta-toxin bound to the cell membrane was solubiized in sodium dodecyl sulfate-ethylenediamine tetraacetic acid mixture (1 % SDS; 20 mM EDTA) and counted in a gamma counter.
Human
Monocyte
Membrane
Gangliosides
67
RESULTS Binding
Experiments
‘251-delta toxin bound to freshly isolated human monocytes. As shown in Figure 1, the binding was proportional to the number of NSE cells per well (between 5 x 10 and 1 X 106) and could be displaced by adding simultaneously an increasing amount of unlabeled toxin. It is worth noting that 95% of the binding was specific, and that the nonspecific binding binding reached a plateau after
was unrelated to the cells. At room temperature 40 mm of incubation (Fig. 2) and was saturable
the (Fig.
3). In experiments in which the unbound radiolabeled delta-toxin was removed after 1 hr of incubation, and the cells were washed, the associated radioactivity remained constant up to 18 hr, suggesting the absence of dissociation of bound ‘25I-delta-toxin from cell membrane (data not shown). Specific binding could also be obtained with cultured monocytes; displacements of l2SI..deltatoxin binding by low amounts of unlabeled toxin were greater using cultured cells rather than freshly isolated monocytes (Table 1). The specific binding (expressed as cpm) was mainly inhibited by preincubation of ‘25I-delta-toxin with GM2 when 0.1 tg gangliosides were used (Table 2); GMI possessed also a significant inhibitory activity when a higher amount (1 sg) was used, whereas GM3 had no significant inhibitory activity at the tested amounts. c pm Id3
.‘o-
A
B
5
4
a
U
3
2
a
S.1O
IS’
0
IS’
2.15’
2.15’
NS(
50
75
Amount
tOO
of unlabeled
150
200
ng
delta-toxIn
Fig. 1. Specific binding of ‘251-delta-toxin to human monocytes. A) Inhibition of binding of 1251-deltatoxin (2 ng; 99,800 cpm) by increasing amounts of unlabeled delta-toxin, on freshly isolated monocytes (106 NSE cells input/well; binding assay: 1 hr at 22#{176}C.B) Binding of ‘251-delta-toxin (2 ng) as a function of increasing number of input NSE + cells per well in the absence (A) or in the presence () of 100 ng unlabeled delta-toxin. N.B. As shown in A, a 48% binding inhibition was obtained with 10 ng of unlabeled delta-toxin on freshly isolated monocytes. When the experiment was performed the following day, similar amount of unlabeled delta-toxin led to an 85% inhibition on 24-hr cultured cells [see Table I].
68
Cavaillon
et al
cpm a
a U 4
0
a
I
/r/
/(‘ #{149}_.__#{149}
2s
s
25
a
so
12.5
mis.
Time Fig. in
2. the
Binding absence
binding
(
of (A)
‘25l-delta-toxin or
in
the
(2
ng)
presence
to
106
(#{149}) of
100
input ng
NSE
cells/well
unlabeled
at
delta-toxin;
22#{176}C as i
a function
symbols
of
represent
time,
specific
cpm).
cpa 1.11
‘4
12
a
a IS
I U
S
4
4/ i Amount Fig.
3.
Binding
amounts
of
toxin.
symbols
TABLE!. Freshly Amount unlabeled delta-toxin
None 2 ng
5 10 20 50
of
‘251-delta-toxin
radiolabeled
to
delta-toxin
represent
of
the
in specific
106 the
i
radlolabeled input
NSE
absence
binding
(A) (
o
ii
5,
delta.toxln cells/well or
in
the
at
22#{176}C as
presence
(4)
a of
function 600
ng
of
increasing
unlabeled
delta-
cpm).
Competition Experiments Between WI-Delta-Toxin Monocytes and Cultured Cells
and
Unlabeled
Delta-Toxin
on
Isolated of
Freshly cpm
5,969 5,047 4,812 4,070 1,394 364
±
SD
± 413 ± 180 ± 227 ± 312 ± 228 ±16
isol ated
monocytes % displacement
15 19 32 77 94
24-hr cpm
±
5,797 ± 3,940 ± 3,210 ± 2,237 ± 1,054 ± 359±80
cultur ed monocytes
SD
899 590 303 125 38
% displacement
32
45 61 82
94
Human TABLE Human
2. Inhibition Monocytes
Monocyte
by Gangliosides
Membrane
of S pecific
Binding
4.5
Inhibitors None 0Ml#{176}1
g
l.0tg GM2O.l jg l.0g 0M3#{176}1
g
l.0g
Cytotoxicity
Gangliosides of
+
2,236
±
332
447
±
1,901 1,285 1,527 886 2,017 2,316
± ± ± ± ± ±
109 158 157 107 273 195
439
±
119 31
767 552 583 495 537
± ± ± ± ±
54 73 24 31 73
on Human
Delta-Toxin
to Fr eshly
I solated
ng
4.5 ng I-#{244}
of Delta-Toxin
1251.
69
150 ng b
Adherent
cpm 1,789 1,462
% inhibition -
18
518
71
975 303 1,552 1,779
45 83 13 I
Cells
As judged by eosine exclusion the previous observation with rabbit
under microscopic observation and contrary to macrophages [2], no toxicity of delta-toxin on human adherent cells could be observed after 1 hr incubation at 37#{176}C with doses as high as 2 jig. However, a cytotoxic effect was obtained on adherent cells that have been cultured for 48 hr when the toxin was present throughout the culture period. The cytotoxic effect was corroborated by a dose-dependent decrease of ‘4C-leucine uptake by the cultured adherent cells (data not shown); maximum inhibition also occurred at the 48th hour of culture.
DISCUSSION
Various bacterial toxins bind to cell membranes through specific recognition of the polysaccharide moiety of the glycosphingolipids. It was previously shown that Clostridium perfringens delta-toxin bound to the GM2 ganglioside or a GM2-like structure present on sheep erythrocytes [11] and on rabbit leukocytes such as alveolar and peritoneal macrophages [10], whereas delta toxin did not bind to rabbit thymocytes [10], which lack GM2 ganglioside on their surface [8]. Thus, delta-toxin can be used for probing the presence of GM2 ganglioside or a GM2-like structure on cell membrane, as other studies use monoclonal antibodies to identify membrane markers. Since very little is known about the ganglioside composition of human monocytes, we have extended our previous work on rabbit macrophages to freshly isolated human monocytes and cultured monocytes that acquire a macrophage-like morphology. Care was taken to avoid platelet contamination since delta-toxin binds to human platelets under these conditions (unpublished observation). We found that delta-toxin binds specifically, saturably, and irreversibly to freshly isolated human monocytes. It is worth noting that 95% of the binding was specific, and that nonspecific binding was unrelated to the cells, since it remained constant whatever the number of input cells (Fig. 1B). Specific binding of delta-toxin could be also achieved on cells maintained in culture up to 72 hr. The main difference with freshly isolated monocytes was a higher competition with low amount of unlabeled toxin, suggesting some modifications of the accessibility or of the affinity of the toxin for its binding site. Preincubation of ‘251-labeled delta-toxin with gangliosides led to a decrease of the specific binding. The inhibition could be observed with GM2 and with GMI, but
70
Cavaillon et al
was insignificant with GM3. erythrocytes [11] and rabbit proved highly more inhibitory as
effective
stronger
as with
GM2
0M2’
for when
In contrast to our previous observations with sheep peritoneal and alveolar macrophages for which GM2 than GMI, in the present study, the latter was almost
specific
binding
gangliosides
inhibition.
were
However,
employed
at low
the
inhibition
concentration
was (0.1
jtg/
assay). As with most studies involving inhibition experiments with gangliosides [3,14,16,22], a certain cross-reactivity was noted, which was certainly due to close molecular structures among this family of glycolipids. Further experiments will be necessary to elucidate the exact nature of the monosialoganglioside(s) present on human monocyte membrane. Furthermore, in contrast to the observations with sheep erythrocytes and rabbit macrophages, delta-toxin cytotoxicity could not be observed within 1 hr at 37#{176}C when studying freshly isolated or cultured human monocytes. In fact, despite the fact that binding occurred within 40 minutes at 22#{176}C,cytotoxicity only took place a few days later. The elucidation of the nature of membrane gangliosides and that of the receptors for these glycosphingolipids among immunocompetent cells will provide information for a better understanding of cellular interactions that often require cell-to-cell contact. Receptors for GM2 have been described on rat alveolar macrophages and to a lesser extent on peritoneal macrophages [21], and receptors for GMI were reported on human peripheral blood mononuclear cells [25]. One can speculate that such receptors are involved
in the
immunomodulation
induced
by exogenous
gangliosides,
such
as
the decrease of mitogen-induced mixed lymphocytes reactions
proliferative responses [14,22,25], the decrease of [25], or the inhibition of an interleukin-2-dependent growth [16]. Interestingly in this last report, as well as in the
cytotoxic T-lymphocyte study of the inhibition of Con shown
A-induced
proliferation of mouse
to be one of the most potent inhibitors. Whether similar gangliosides, associated
thymocytes,
within the cell membrane,
GM2
was
can be as
efficient as the exogenous gangliosides used in these experiments remains an open question. In this context, the inducibility of such membrane markers by different mechanisms may appear as an important factor. Indeed, it has already been shown that the in vivo activation of macrophages, by thioglycolate medium, BCG, or Corynebacterium parvum for example, led to the appearance of a distinct GM1 species [15] or to an increase of the asialo-G1-positive cells [1,26]. The latter marker was also increased on spleen leukocytes of lymphocytic choriomeningitidis-virus-infected mice [27]. Similarly, the expression of new membrane markers during cell maturation triggering
[6,19]
may This
reflect is the
surface of monocytes
human
and
acquisition of new functions [23]. first evidence for the presence of a monosialoganglioside adherent macrophage-like
toxin suggests a reorganisation maturation to macrophage, activity
of the
injection incubation
toxin.
The
of delta-toxin of oocytes
cells.
The glycolipid appears to cells surfaces, but the delayed
of such or a decrease second
structure within of intracellular
hypothesis
into Xenopus with toxin was
is highly
laevis oocytes lytic (unpublished
be
the cell resistance
improbable did not lead observation).
on
present cytotoxic membrane to the since
the
on both effect of during cytotoxic
intracellular
to lysis,
whereas
ACKNOWLEDGMENTS The Mariuzza National
authors
thank
Ms.
L.
Cayrol
for
typing
the
manuscript
and
for linguistic advice. We thank Mrs. Cristofari and her colleagues de Transfusion Sanguine) for providing the blood samples.
Dr.
Roy
(Centre
Human
Monocyte
Membrane
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