Anaphylactic properties of monohaptenic dinitrophenylated tripalmitoyl-S-glycerylcysteinyl lipopeptides

Molecular Immunology, Vol. 27,No. 3,pp.241-245,1990 Printed in Great Britain.

Ol61-5890/90 $3.00+O.OO IQ 1990PergamonPressplc

ANAPHYLACTIC PROPERTIES OF MONOHAPTENIC DINITROPHENYLATED TRIPALMITOYL-S-GLYCERYLCYSTEINYL LIPOPEPTIDES CONRAD H. SCHNEIDER,* HANSPETERROLL],* J~~RGMETzGERt and GUENTHERJUNG? *Institute for Clinical Immunology, University of Berne, Inselspital, CH-3010 Berne, Switzerland and TInstitut fiir Organische Chemie der UniversitLt Tiibingen. Auf der Morganstelle 18, D-7400 Tiibingen, F.R.G.

(First received 26 January 1989; accepted in revised form 27 July 1989) Abstract-Tripalmitoyl-S-glycerylcysteinyl lipopeptides are B-cell and macrophage activating and may be used as low molecular weight immunogens of considerable potency and even as vaccines when conjugated with suitable epitopic structures. Selected lipopeptides carrying single Dnp haptens were found to evoke mild passive cutaneous anaphylaxis in guinea pigs sensitized against Dnp. The reactions were observed after intravenous injection whereas intradermally applied antigen was negative. The anaphylactogenicity seems unrelated to micelle or aggregate formation of the insoluble peptides which require lecithin additions as well as sonication to become solubilized. The dinitrophenylated lipopeptide tripalmitoyl-S-glycerylcysteinyl-seryl-lysine produced toxic reactions which were not observed with the lipopeptide devoid of Dnp. Dinitrophenylated tripalmitoyl-S-glycerylcysteinyl-1,6-diaminohexane and tripalmitoyl-S-glycerylcysteinyl-lysine did not show these toxic reactions

It was of interest to study anaphylactogenic properties of selected conjugates from this lipopeptide category and to compare the data with those previously obtained with soluble monohaptenic anaphylactogens.

INTRODUCTION Monohaptenic antigens that elicit histamine release from vasoactive cells are a heterogeneous group. They include glycopeptide conjugates (Guenin and Schneider, 1983), penicillin antigens (Ueno et al., 1984; Guenin and Schneider, 1984~) as well as antigens with certain hydrocarbon auxiliary groups (Raffel, 1973; Schneider et al., 1985). These compounds are all soluble in aqueous media and do not specific antisera. Their precipitate anti-hapten efficacy thus is not based on simple aggregation which produces functionally oligohaptenic units capable of cross-linking the antibodies on vasoactive cells. Recently, defined and effective immunogens were obtained by coupling peptidic and other single haptens to B-cell and macrophage activating tripalmitoyl-S-glycerylcysteinyl lipopeptide carriers (Jung et al., 1985; Bessler et al., 1985). In particular, a fully protective and totally synthetic low molecular weight vaccine against foot-and-mouth disease virus was obtained by covalent attachment of tripalmitoyl-Sglycerylcysteinyl-seryl-serine to the virus protein segment VP1 136-154 (subtype 0, k) (Wiesmiiller et ul., 1988; Krug et al., 1990). The lipopeptide derivatives are rather insoluble in aqueous solvents and require for solubilization the addition of lecithin as well as ultrasonic treatment. However, they incorporate immediately into the lipid bilayer of cell membranes and liposomes as shown by biophysical studies (Metzger, 1988).

MATERIALS AND METHODS Dinitrophenylated

Four lipopeptides containing the lipoamino acid Pam,Cys-OH (Wiesmiiller et aI., 1983) at the Nterminus (Fig. I) were prepared and dinitrophenylto give: ated with I-fluoro-2,4-dinitrobenzene Pam,Cys-HMD-Dnp (HMD: 1,6_diaminohexane), 1204.8 g/mol; 1174.8 g/mol; Pam,Cys-Lys(Dnp), Pam, Cys-Ser-Lys(Dnp), 129 1.8 g/mol; Pam, Cys-SerLys-[Lys(Dnp)], trifluoroacetate, 2122.55 g/mol. The first three conjugates are monovalent with respect to Dnp; Pam,Cys-Ser-Lys[Lys(Dnp)1, trifluoroacetate is trivalent. The conjugates were purified on Sephadex LH-20 and characterized by amino acid analysis, “C-NMR and field-desorption mass spectrometry. Details of the syntheses and characterizations are to be presented elsewhere. PAL conjugates The decalysine carrier peptide PAL containing four adipic acid substituents on lysine side chains (Rolli and Schneider, 1987) was substituted by involving the six remaining free lysine t-amino groups and the Butaz hapten (Schneider ef al., 1987) or l-fluoro-2,4dinitrobenzene. The conjugates Butaz, -PAL and Drip,-PAL were isolated by gel filtration through Sephadex G-50.

Correspondence: Prof. C. H. Schneider, Institute for Clinical Immunology,

Inselspital.

CH-3010

lipopeptides

Berne. Switzerland. 241

242

CONRAD

CH~-(CH~)~L

H.

SCHNEIDER~~ al

serum, again sonicated and then incubated at 37 C for 1 hr and overnight at 4°C. After centrifugation and decantation in the cold, the tubes were washed twice with 2ml PBS and once with HzO. To each tube, 2 ml 0.1 M NaOH was added in order to dissolve the protein. Aliquots of 0.3 ml of the NaOH solution were then used for measuring Folin-Lowry colour. A slight turbidity of the final solution did not interfere with the determination of the absorbance at 750 nm in 1 cm cells.

-CO-0-7H2

CH3-(CH2),L-CO-O-~H CH2 I S

I

CH~-(CH~),L

CH2 I -CO-HN-CH - COOH

Fig. 1. Structure of the lipoamino acid N-palmitoyl-S-[2,3bis(palmitoyloxy)-(2RS)-propyl]-[RI-cysteine: abbreviated Pam,Cys-OH. Benzoyl-Lys(Dnp) This was prepared (1963). Anti-Dnp

antiserum

according

to Amkraut

et al.

CT-26

The antiserum was obtained by immunizing rabbits with bovine gamma globulin carrying approx 50 Dnp haptenic groups. A standard schedule involving Freund’s complete adjuvant was used (Schneider et al.. 1979). Passive cutaneous anaphylaxis

(PCA)

The technique of Watanabe and Ovary (1977) was essentially followed. Out-bred guinea pigs of 2.50-300 g were injected i.d. with selected dilutions of antiserum in 0.01 A4 phosphate buffered saline pH 7.4 (PBS). After 18-20 hr, blueing reactions were elicited by iv. injection of 0.5 ml aqueous Evan’s blue together with the conjugate in 0.5 ml PBS. Alteranimals were injected i.v. with 1 ml natively, antiserum dilution and 20 hr later with 0.5 ml Evan’s blue in PBS. Blueing reactions were obtained by i.d. injections of conjugates in 0.1 ml PBS immediately before Evan’s blue administration. Quantitation of the blue areas was identical in both versions of the test and is obtained by measuring two perpendicular diameters. The time for full development of the reaction was usually 20 min.

RESULTS

Immunoprecipitation Figure 2 shows that Pam?Cys-Lys(Dnp) is unable to precipitate anti-Dnp antiserum over a considerable concentration range within which Drip,-PAL readily precipitated antibody. Protein precipitation sets in only at high concentration, but the effect appears unspecific since sera from non-immunized rabbits also showed this precipitation. These data suggest that formation of micellar units which is expected to occur upon sonication in the presence of lecithin is slight or that the units formed are ineffective in displaying a stable and accessible oligohaptenic arrangement which of course would be a prerequisite for immunoprecipitation. Anaphylactogenicit), After intravenous injection, the monohaptenic Dnp-derivatives Pam,Cys-HMD-Dnp and Pam,CysLys(Dnp) clearly evoked cutaneous anaphylactic reactions in guinea pigs passively sensitized by anti-Dnp antiserum (Table 1). The reactions are no less than those obtainable with soluble benzoyl-Lys (Dnp), a compound belonging to a well-described category of monohaptenic anaphylactogens (Guenin and Schneider, 1984b). Lecithin which is present as a solubilizer (cf. “Solubilizations”) has no effect; it also

Solubiltations The aqueous solvent generally used for the insoluble Dnp antigens was PBS with 2 mgjml of soybean lecithin (PBS-Let); omission of lecithin is specifically indicated in these cases. For solubilization the mixtures were subjected to ultrasonic treatment in small vessels kept in an ultrasonic bath for several minutes until clear to slightly turbid solutions were obtained. This treatment occurred immediately before use, e.g. before intravenous or intradermal injections in the PCA experiments. Immunoprecipitations Aliquots of 0.5 ml PBS-Let containing graded amounts of Dnp-antigen were sonicated in small glass tubes, mixed with 0.5 ml antiserum or normal rabbit

0’

/

2

8

Haptenic

32 128 concentration

512 2048 (pmol/l)

Fig. 2. Immunoprecipitation of anti-Dnp antiserum with antiserum CT-26 precipitated Dnp-antigens; A-A: with Drip,-PAL: O-0: antiserum CT-26 precipitated with Pam,Cys:Lys(Dnp); m-m: normal rabbit serum precipitated with Pam,Cys-Lys(Dnp). Sera from three unimmunized rabbits w&e sinily tested giving virtually identical results.

Anaphylactogenic lipopeptides Table I. Elicitation

of PCA by Dnp-derivatives /lmol injected

Derivative

per animal

iv.

Pam,Cys-HMD-Dnp Pam,Cys-Lys(Dnp) Pam,Cys-Ser-Lys(Dnp)

Number of animals

I.0 1.0 1.0

3 5 1 3 3 2 1 3 4 3

0.1 1.0 0.1 I.0

Pam,Cys-Ser-Lys-[Lys(Dnp)], Benzoyl-Lys(Dnp) Benz&Lys(Dnp) PBS-Let

in guinea pigs intradermally

in PBS-Lee in PBS

I .o -

l/IO 13-20 ORb

OR oeg

Table 2. Elicitation

Pepude l.O~mol, i_l/, Pam,Cys-Ser-Lys Pam,Cys-HMD,HCI “The number

MlMM 27*3--D

of PCA by lipopeptides

devoid

of hapten

Sensitization id.

Number of positive blue sites versus total number of sitesa

CT-26; NS; CT-26; NS;

0.24 O/l8 Oil8 o/12

l/IO l/IO I,‘10 l/IO

of sites per guinea pig was 6: NS: normal

serum.

sensitized

by anti-Dnp

antiserum

CT-26

Mean diameter of blue areas in mm (range) sensitized by antiserum diluted: 1120 1140 I /SO I1160 11320 11640 1 I-13 l9-OR GB GB oeg GE 10 28-OR 19-OR

7-10 13-OR 20 GB neg GB 5 20-OR 19-OR

neg 12-22 10 GB neg GB 4 19-OR 12-22

neg neg- I 7 GB GB neg GB 4 14-18 neg-2 1

oeg neg- 10 GB GB neg GB

neg

neg

neg

oeg

neg

“NS: site injected with a l/IO dilution of normal rabbit serum. bOR: Over-reaction; neg: no blueing; GB: general blueing of large parts of the animal,

did not significantly affect the potency of benzoylLys(Dnp) which was tested in the presence and absence of lecithin. The trihaptenic Pam,Cys-SerLys-[Lys(Dnp)], evoked general blueing which may be interpreted as over-reaction of a potent anaphylactogen and/or a non-specific cytotoxic reaction. It gave on the other hand specific, measurable reactions at the lower 0.1 pmol dose. Pam, Cys-Ser-Lys(Dnp) produced general blueing of the animals and no specific reactions could be measured at the sites of injected antiserum or normal serum with two exceptions, but at the lower dose of 0.1 pmol it was clearly negative. At the higher dose the animals also showed symptoms which ranged from restlessness to difficulty of breathing. Not shown in Table 1 are two animals that underwent exactly the schedule of the table but were i.d. injected with dilutions of normal rabbit serum instead of antiserum. Upon i.v. injection of 1.0 pm01 Pam,CysSer-Lys(Dnp), genera1 blueing occurred as well as temporary unconsciousness. The lipopeptide without the Dnp hapten, Pam,Cys-Ser-Lys was then tested (Table 2) and found to evoke no reactions at all. It is thus evident that in addition to possible but unobservable PCA, toxic reactions may occur with Pam,Cys-Ser-Lys (Dnp) but not with the lipopeptide devoid of Dnp. Although the sites injected with normal rabbit serum were generally negative, it seemed desirable to further confirm the specificity of the reactions elicited by Pam,Cys-HMD-Dnp and Pam,Cys-Lys(Dnp). Table 3 shows that in addition to normal serum also an antiserum with the irrelevant anti-Butaz specificity gave injection sites which remained unresponsive when either of the Dnp-antigens were administered.

243

no quantitation

oeg IO-18 neg-IS

negy7 GB GB neg GB oeg neg-9 neg- I 4 nee

NS”

w neg GB GB neg GB neg oeg oeg neg

possible.

However, when homologous hexavalent Butaz,-PAL was subsequently injected, the anti-Butaz sites became highly responsive, thus constituting a strong positive control. Table 4 deals with experiments where intravenously sensitized guinea pigs were injected intradermally with Dnp-antigen. It is evident that up to doses of 2 nmol mostly negative responses are observed. At the high doses of 20 and 200 nmol, Pam,Cys-Lys(Dnp) elicits clear reactions in about half the animals of a group of nine. However, similar reactions were also obtained in animals injected with normal serum. These manifestations are therefore essentially non-specific. It is noteworthy that the high reactor animals also showed slight general symptoms, i.e. restlessness and beginning difficulty of breathing shortly after antigen administration. This applies not only to the CT-26-treated animals but also to animals Nos 18 and 19 which received normal serum. Omission of lecithin had hardly a significant effect. The data essentially indicate that Pam,Cys-Lys (Dnp) which is anaphylactogenic when given by the intravenous route does not evoke specific anaphylattic reactions at the usual doses when injected intradermally. DISCUSSION

The tripalmitoyl lipopeptide conjugates studied here are not soluble in aqueous media and were sonicated in the presence of lecithin immediately before use, to give clear to slightly turbid suspensions. At lOO-fold magnification, droplets of lecithin and small solid fragments of the lipopeptides are still visible at this stage. After a few minutes substantial precipitation becomes observable suggesting instability of the solubilized systems. It should be kept in mind however, that after injection into an organism, rapid transport of finely divided material to cell membranes sets in, where true molecular solubilization may become possible. Micellar units that may also form during sonication seem of little importance in the present experiments because intradermal injection of Pam,Cys-Lys(Dnp) did not evoke specific reactions (Table 4). Micelles of some stability and

CONRAD H. SCHNEIDER et al.

244 Table 3.Elicitation

Derwatwe

i Y.

(pm”11

of PCA m gumea pigs intradermally antise&

Mean diameter of blue area in mm I” sites sensitized by anti-Butaz: I,‘10 NS: CT-26: I,20

Animal No.

I

Pam,Cys-HMD-Dnp

sensitized by CT-26 and anti-Butaz

(1.0) Pam,Cys-Lys(Dnp) (1.0) Butaz-PALh (0.04 /~rnol Butaz) 4

I .I0

II I3 19 OR

“Q *eg “e-z “e.E

“eg “W “eg neg

I2 13 I9 OR

22 OR 23 22

“Q “Q “% nee

‘For abbreviations see footnote to Table I. h Immediately after readmg the PCA reactions of lines 14. we injected the Butaz,-PAL the animals were aga” read after another 20 min.

displaying a multitude of accessible haptenic groups would be expected to cross-link antibodies on the mast cells and thus initiate localized cutaneous anaphylaxis. Micelle contribution was also absent in the immunoprecipitation experiments where Pam, CysLys(Dnp) did not achieve specific precipitations. On the other hand, Pam,Cys-Lys(Dnp) and Pam,Cys-HMD-Dnp clearly evoked anaphylaxis after i.v. injection into guinea pigs passively sensitized by intradermal anti-Dnp antiserum. This behaviour. namely anaphylactic efficacy in guinea pigs after intravenous, but not after intradermal injection, has been found before within a group of monohaptenic anaphylactogens which carry next to the hapten rather well-specified hydrocarbon auxiliary groups (Schneider et al., 1985; Schneider et ul.. 1982). The auxiliary group may contain hydrocarbons such as the benzene ring or short aliphatic chains as well as carboxyl groups in selected positions. The present lipopeptides transcend the dimensions of the known monohaptenic anaphylactogens containing a hydrocarbon auxiliary group, because their three palmitoyl residues CH,-(CH,),,-COrequire considerable space. Pam,Cys-Lys(Dnp) carries a free carboxyl group, but Pam,Cys-HMD-Dnp has none. It is important to note from Table I that the carboxyl-bearing conjugate was a markedly better anaphylactogen than the HMD derivative. This result is in line with experiences within the group of the above monohaptenic

anaphylactogens (Guenin and Schneider, 1984~ and 19846) and could indicate that the palmitoyl lipopeptides are acting via the same, as yet undefined. anaphylactogenic mechanism. In any case, it will be important to recognize that soluble as well as finely divided insoluble monohaptens may initiate anaphylactic reactions under apparently quite similar conditions. In addition to anaphylactogenicity. non-immunospecific manifestations were noted in some instances. This was particularly the case after i.v. injection of Pam,Cys-Ser-Lys(Dnp) into sensitized and nonsensitized, rabbit serum-treated animals, where general blueing as well as systemic reactions occurred. On the other hand. the lipopeptide Pam,Cys-Ser-Lys lacking Dnp was negative Pam,Cys-Lys(Dnp), which gave clearcut immunospecific reactions after intravenous injection (Table I), also produced unspecific blueing at high intradermal doses (Table 4). It appears that these well-documented unspecific reactions are not simply due to the lipopeptide carriers as such but rather to Dnp or to the combination of Dnp plus carrier. With regard to the use of the tripalmitoyl conjugates as immunogens, it should be noted that Pam,Cys-HMD-Dnp or Pam,Cys-Lys(Dnp) arc anaphylactogens with a potency well below that of multivalent antigens. This will be an asset in immunizations where specific IgE or other anaphylactic antibodies may become involved, because in this

Table 4. Elutatmn of cutaneous anaphylaxis by Pam,Cys-Lys(Dnp) after intravenous paswe tratm” of anrl-Dnn antiserum CT-26 or normal serum from the rabbit Diluted serun, I.\ (1_~ ml) Pam,Cq\-Lys(Dnp)

4 CT-20

Pam,Cq\-Ly\(Dnp) \\lthout leathin Pam,C)+L>\(Dnp)

4 CT-26

Drip,-PAL

I 4 NS

I .4 CT-26

Ammal NO

I~ 3 4 56 7 x9 IO I2 Ii 14~15 16 IX 19 20-22

“Dependmg on the animal. mjectlon of PBS-Let up to 4mm This effect has been wbtracted

and

admima-

Mean diameter” of blue areas in mm (range) after an elicitor dose I” nmol of 0.2 20 LOO 0.02 20 “eg “eg “Q “Q? 4-5 “Q7 “V “eg neg neg 6 IO

neg “V “eg neg 4-5 “eg “eg “V “eg 2 9 IO

“E-g “eg “eg 7 3 5 “ee 7 “eg neg.3 2 II -I2

nsg neg 3 .c I0 h-10 24 5 “rp 76 8 I? 15

neg 4 X-IO I? II-I? 67 7 neg-3 3 8 II

or PBS produces a sharp blue point or ii blue area of from the actually measured values.

Anaphylactogen ic lipopeptides situation the multivalent antigen would evoke more pronounced anaphylaxis. It appears even ible that adequate immunizing doses can be lished which will be low enough to allow none reactions described here to come into play.

much possestabof the

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