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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