phosphate-buffered saline;. RE\. A, reticuloendotheliosis-associated virus;. RSV,. Rous sarcoma virus;. T1 protein, transmembrane envelope protein. Throughout.
Synthetic hexapeptides derived from the transmembrane envelope proteins of retroviruses suppress N-formylpeptide--induced monocyte polarization Robert and
Rik
Oostendorp J. Scheper*
Wim
A.J.
5Department
of Pathology,
Immunology,
Central
Free
Veterinary
M.
University
M. Schaaper1
Hospital,
Institute,
Lelystad,
Amsterdam The
Abstract: Retroviral infections are frequently associated with immunosuppression. Retroviral transmembrane envelope proteins (TM proteins) play an important role in this phenomenon. CKS-1 7, a synthetic heptadecapeptide, represents the immunosuppressive site of these retroviral TM proteins. Here we report on the further delineation of this immunosuppressive site using CKS-1 7-derived hexapeptides. The N-formyl-methionylleucyl-phenylalanine-induced monocyte polarization assay was used throughout this study because this monocyte function has been shown to be highly sensitive to TM protein pl5E-related immunosuppression. We found that in addition to CKS-1 7 one CKS-1 7-derived hexapeptide, LDLLFL, reversibly inhibited monocyte polarization, with 50% inhibitory concentrations of 20 and 2 tM respectively. LDLLFL-mediated inhibition was sequence specific because the reverse peptide LFLLDL and scrambled peptides were not inhibitory. Hexapeptides corresponding to LDLLFL, but derived from various retroviruses other than murine leukemia virus, also inhibited monocyte polarization. Peptides most homologous to LDLLFL - LDILFL (feline leukemia virus) and LDLLFW (human T lymphotropic virus types I and II)were the most potent inhibitors. Peptides homologous to primate and human endogenous proviruses were not suppressive. LDLLFL and some of its homologues also inhibited polarization of neutrophilic granulocytes. These findings lend further support to the view that conserved retroviral TM protein-related peptides can play an important role in suppression of inflammatory cell function as encountered in retrovirus-associated immunosuppression. J. Leukoc. Biol. 51: 282-288; 1992. Key
Words:
tides
monocyte
retroviruses
immunosuppression
synthetic
Jacob and
Post,t
Rob
tLaboratory
H. Meloen,t
of Molecular
Netherlands
immunosuppression induced by preparations of LMWF derived from human tumors. Retroviral pl5E was found t be immunosuppressive [38], and a recombinant protein der ived from a murine retroviral pl5E gene [35, 37] showed similar activity. Tumor-derived pl5E-related LMWFs hay been shown to depress delayed-type hypersensitivity reac tions in mice [27, 42]. These results led to the hypothesis tha pl5E-related LMWFs may facilitate tumor progression [38] TM proteins from various retroviruses share homology ii a sequence of 26 amino acids [7]. CKS-17, a synthetic hep tadecapeptide derived from this consensus sequence, in hibited many different in vitro lymphocyte [5, 14, 21, 23, 24 29], natural killer (NK) cell [18], and monocyte [17, 20] func tions. CKS-17-induced immunosuppression was als demonstrated in vivo [26]. More importantly, CKS-V homologues derived from the amino acid sequences of HIV-: and human T lymphotropic virus type I (HTLV-I) showe similar in vitro suppressive activities [4, 36]. These result favor the view that the CKS-17-like sequences represent major immunosuppressive site of the retroviral TM proteins Considering the importance of the CKS-17 domain ii pl5E-related immunosuppression, we decided to investigati this sequence further and find out whether CKS-17-relate immunosuppression might reside in even smaller peptides For this purpose we synthesized a series of overlapping pep tides covering the complete sequence of CKS-17. This so called PEPSCAN approach has proved successful ii delineating antibody binding sites [13] as well as T cell epi topes [48]. We used hexapeptides because it had previousl been shown that retrovirus-related hexapeptides could sup press T lymphocyte responses to interleukin 2 (IL-2) [46]
pepAbbreviations:
function
bumin;
BaEV,
DAE,
dimethyl
baboon
endogenous
1,2-diaminoethane;
sulfoxide;
FeLV,
feline
methionyl-leucyl-phenylalanine;
INTRODUCTION
human
HIV,
Immunosuppression tients [6, 40], positive people
human [43],
is frequently encountered immunodeficiency virus and patients with chronic
in cancer (HIV)-seroinflammatory
pa-
Journal
of Leukocyte
Biology
Volume
51,
March
virus;
M BS, mia
1992
leukemia
virus;
human 50%
limpet
LMWF,
NK
cell,
natural
cell;
protein, codes
envelope
representing
glutamic
L-amino
acid; P, proline;
tryptophan; Reprint University
N,
and requests:
IL-2,
are G,
Q,
asparagine;
RSV,
MuLV,
Rous
Throughout
used:
A,
glycine;
murine
sarcoma this
alanine;
D,
glutamine;
R,
arginine;
lym
leuke
RE\
virus; paper
T1
one-lette
aspartic K,
2
factor, saline;
I, isoleucine;
lysine;
T, threonine;
E
acid; L,
let. ‘v
Y, tyrosine. R.A.J.
Hospital,
Dr
April
1, 1991;
Oostendorp,
Boelelaan
Department of HV Amsterdam,
1117.
1081
July
24,
lands. Received
T
interleukin
phosphate-buffered
protein.
acids
F, phenylalanine;
liqufi
human
low-molecular-weight
PBS, virus;
transmembrane
N-formyl retrovirus
HTLV,
ester;
killer
al
DMSC IMLP,
concentration;
hemocyanin;
serum
high-performance
albumin;
inhibitory
bovine
endogenous
HPLC,
serum
reticuloendotheliosis-associated
cine;
virus; human
maleimido-benzoyl-hydroxy-succinimide
virus;
A,
IC50,
keyhole
KLH,
diseases [30, 44]. Defective monocyte responses to chemotactic stimuli are most frequently noted. It has been demonstrated that such immunosuppression may result from circulating factors antigenically related to retroviral transmembrane envelope (TM) proteins [3, 6, 41, 43, 44]. Both human and murine tumors were found to secrete lowmolecular-weight factors (LMWFs) with immunosuppressive properties [38]. Antibodies directed against the pl5E TM protein ofmurine leukemia virus (MuLV) abolished the 282
photropic
HSA,
BSA,
1,6-diaminohexane;
HERV,
immunodeficiency
chromatography;
virus;
DAH,
accepted
1991.
Pathology, Fred The Nether
Furthermore, we investigated )f homologous hexapeptides Jomains of other retroviral All peptides were evaluated V-formyl-methionyl-leucyl-phenylalanine nonocyte polarization, a :hemotactic responsiveness KS-17-derived Ictivity.
ilso
hexapeptide,
LDLLFL,
shows
hexapeptide
LDLLFL
homologues
Several
found
to
suppress
V1ATERIALS
immunosuppressive activities derived from the CKS-17-like TM proteins. for their capacity to suppress (fMLP)-induced function closely correlated to [40]. Here we report that one
AND
monocyte
any
of the
angular
the
peptides :echnique as eptides were amide xchange
V
Percent
A)
followed
:HPLC and scid analysis. Throughout
by
standard solid-phase and Frank [15]. All group) and carboxyl
were 2-X8,
high-performance
purified BioRad,
liquid
using ionRichmond,
chromatography
verified by reverse-phase HPLC Purity of the peptides varied from this study we used FLEEI (Sigma
and amino 85 to 95%. Chemical
St. Louis, MO) as a negative control peptide. In some experiments the cysteine-containing peptide LDLLFL was coupled to 1,2-diaminoethane (DAE), l,6-diaminohexane (DAH), bovine serum albumin (BSA), or C.eyhole limpet hemocyanin using the maleimido-benzoylsydroxy-succinimide ester (MBS) coupling reagent [22]. CKS-17 coupled to human serum albumin (CKS-17-HSA) Nas a generous gift from Dr. G.J. Cianciolo (Sphinx Biotechologies Corp., Durham, NC). onocytes
and
Monocyte
Background
and monocytes of the monocytes polarization
siveness
of
monocytes [44]. in a 37#{176}Cwater bath 10 ml in culture medium. ice in culture medium (5 mm at 500g), llowed to recover in culture medium for y was >95% as assessed using trypan uots
P
of 200
12-77-mm
human thawed ice-cold
l
containing polypropylene
fBecton-Dickinson
5-tl
aliquots.
0.2
Co., Finally
25
Oxford, jl
of
In
brief, monocytes and diluted slowly After being washed the monocytes were 40-60 mm. Viabilblue exclusion. Ali-
x 106 monocytes
tubes
(Falcon
CA). fMLP
pared
were
Labware,
Peptide (Sigma
Vhackgrouncl
Chemical)
culture
monocytes
was
prepared
not
reduced
com-
monocytes.
Granulocyte
Polarization
ing the same cell and fMLP concentrations. Depending on the donor, 70-85% ofthe granulocytes polarized in response to IMLP. Background polarization was usually 10-30%.
Statistical
Analysis
Data were analyzed using Student’s of < .05 were considered statistically was
repeated
at
least
unpaired significant.
three
t-test. P values Each expeni-
times.
Control
CKS-l7
**
LQNRRG
QNRRGL C.) ‘0 .-
NRRGLD
C.)
RGLDLL
RRGLDL
GLDLLF
*
LDLLFL DLLFLK LLFLKE LFLKEG
FLKEGG LKEGGL
in
0
was
dded, to reach a final concentration of 10 mM. The tubes ‘ere incubated at 37#{176}Cin a water bath for 20 mm. The inubation was stopped by addition of 250 l of ice-cold 10% nrmaldehyde in 0.05 M phosphate-buffered saline (PBS) pH 7.2). The cells were kept at 4#{176}C until counting in a hemoytometer using an ordinary light microscope. The test was andomized and read “blindly” by two people; 200 cells were ounted from each tube. A cell was considered polarized if
Oostendorp
monoV,i,edjtim
Human gnanulocytes were obtained from buffy coats, using the pelleted cells from density centnifugation through FicollHypaque (Pharmacia, Uppsala, Sweden). Residual erythrocytes were lysed in ice-cold carbonate-buffered ammonium hydroxide solution. The cells were kept on ice and washed in ice-cold medium thereafter. Granulocyte polarization was then assayed as described under monocyte polarization, us-
added added
and
of polarized compounds, that with
only. Depending on the donor, polarized in response to fMLP. was usually less than 3%. Respon-
stored
of freshly
Granulocytes
division
was
frozen
to that
ment
Polarization
uman buffy coats were obtained from healthy volunteers aged 20 to 35 years). Human monocytes were obtained from uffy coats by an elutriation centrifugation technique [1]. onocytes were generally more than 95% pure as assessed y morphology and nonspecific esterase staining [24]. onocytes were cryopreserved [19] and stored in 10% imethyl sulfoxide (DMSO) (Merck, Darmstadt, FRG) in ulture medium RPMI 1640 (Gibco, Paisley, UK) with 25 M HEPES, 2 nM L-glutamine, 10% fetal calf serum (Hylone Laboratories, Logan, UT). The monocyte polarization assay used here was performed 5 described previously using elutriator-purified and ryopreserved ere rapidly
V1)ackgrouti(l
-
______________________ ) x 100
-
in which V1,0 represents the percentage cytes in the presence of fMLP and test that with IMLP only, and VI)ackgroun(l medium
Peptides (AG
= (1
inhibition
V fl5e(liutfl
METHODS
group) termini. chromatography
formula:
were
polarization.
were synthesized by a modified by Hagenmaier modified at amino (acetyl
following
broadened
suppressive
Synthesis
Fhe
polarized
characteristics showed: elongation, tnlamellipodia [30, 43]. cells was converted to percent inhibition
Percent using
25-40%
Peptide
following
shape,
20
40
% Inhibition Fig.
1. Effects
acid
sequence
monocyte 50
Bars Routinely
Statistics
as
