Clin Exp Immunol 1993; 93:120-125

The induction of cell-associated and secreted IL-1 by iscoms,matrix or micelles in murine splenic cells

M. VILLACRES-ERIKSSON*, M. BERGSTROM-MOLLAOGLUt, H. KABERGt, K. LOVGREN* &B. MOREIN*t *Swedish University of Agricultural Sciences, Department of Veterinary Microbiology, Division of Virology,

and tThe National Veterinary Institute, Department of Virology, Uppsala, Sweden

(Acceptedfor publication 17 March 1993)

SUMMARY

The kinetics of the expression of membrane-associated IL-1 (mIL-l) and soluble IL-1 (sIL-l) was

studied in in vitro stimulated spleen cells from non-primed mice or from mice primed with influenzavirus antigens incorporated in the immuno-stimulating complexes (iscoms) or as micelles. Matrix,which is the carrier structure for the antigens in the iscom, was used as a non-antigen stimulus. TheIL-I produced was assayed in an IL-l -dependent cell line and the specificity was demonstrated in a

blocking experiment with antiserum to IL-lo. Soluble IL-lot was also quantified in ELISA. Iscomsand matrix induced production ofmIL- I and sIL- I in cultures from non-treated mice as well as frommice primed 4 days before with iscoms or micelles. Micelles were a less strong stimulus and did notinduce production of sIL- 1. Micelles induced production ofmIL- in cultures from non-primed miceor from mice which were recently immunized with micelles. No mIL-1 expression was induced bymicelles if the spleen cells originated from mice immunized shortly before with iscoms. Depletionexperiments demonstrated that sIL-l was produced by adherent cells upon stimulation with iscomsor matrix. However, factor(s) from the non-adherent cells seem to be necessary for optimal secretionof sIL-l.

Keywords iscom Quil A IL-1 influenza virus antigen presentation

INTRODUCTION

An early product of antigen-presenting cells (APC) which playsa major role in the initiation of the immune response is IL-I[1,2]. IL-1 is expressed and produced by monocytes andmacrophages, as well as some other cell types [3]. Particulatestimuli such as heat-killed Listeria bacteria seem to induce IL-Iproduction more efficiently than soluble proteins [4]. IL-ldisplays a broad variety of biologic activities, reflecting a criticalrole in the initiation of the immune response as well as ininflammatory reactions (reviewed in [5]).

Intensive efforts are being made to develop adjuvants forefficient vaccine formulations. Several adjuvant active sub-stances stimulate IL-1 production, e.g. lipopolysaccharide(LPS), muramyl dipeptide (MDP) [6] or cholera toxin [7]. Anefficient system for antigen presentation, initiation and stimula-tion of immune responses is the immuno-stimulating complex(iscom). The iscom is a cage-like structure containing antigen ina multimeric form with a built-in adjuvant component [8,9].Iscoms have been shown to elicit a strong immune response even

Correspondence: Maria Villacres-Eriksson, Swedish University ofAgricultural Sciences, Department of Veterinary Microbiology, Divi-sion of Virology, Box 585, Biomedical Center, 75123 Uppsala, Sweden.

to poorly antigenic molecules such as bovine serum albumin(BSA) [10], and small peptides [11]. Strong humoral and cell-mediated immunity is induced by antigen presented in iscomparticles (reviewed in [12]). Recent studies have shown thatiscom-borne antigen is able to induce CD8+ cytotoxic Tlymphocytes (CTL) under MHC class I restriction [13-16].Mechanisms contributing to the efficient induction of immuneresponses by iscoms may involve a number of events, e.g. theattraction of neutrophils to the injection site [17]. Also,increased numbers of cells bearing MHC class II molecules wereobserved after treatment with iscoms [18,19]. In mice immu-nized with iscoms, memory T cells are recruited in an antigen-specific manner, producing IL-2 and interferon-gamma (IFN-y)upon restimulation [20,21]. These effects and the transient highspontaneous proliferation of murine splenocytes cultured withmatrix or iscom [20] may be partly due to induction of IL-I;these results led us to address the question ofthe effect of iscomsor matrix on APC and their secretion of monokines. Theexperiments reported here were designed to give insight into thecapacity of iscoms or matrix to induce IL-I in spleen cells (SC),the effects of the presence of memory cells on the level of IL-Iexpressed, the nature of the IL-I response, and the phenotype ofthe responding cell populations.

120

Induction of IL-I by iscoms

MATERIALS AND METHODS

Preparation of iscoms and micellesIscoms and micelles were prepared to contain the haemagglu-tinin (HA) and neuraminidase (NA) envelope glycoproteinsfrom influenza A virus HINI (PR/8/34) as described [22].

Preparation ofmatrixMatrix, i.e. the carrier structure of iscoms composed ofcholesterol, phospholipid and Quil A, was prepared as describedbefore [23]. The matrix and iscoms were characterized morpho-logically by electron microscopy, and the S value was measuredas described for iscoms [23].

ImmunizationsGnotobiotic BALB/c mice 10-12 weeks old (National Veterin-ary Institute, Uppsala, Sweden) were used in all experiments.Mice were immunized twice 4 weeks apart subcutaneously with1 pg influenza virus iscoms (n = 5) or micelles (n = 4). Four daysafter the last immunization the mice were killed and single-cellsuspensions were prepared from the spleens. Non-immunizedmice were used as negative controls (n = 5).

In vitro stimulation ofSCfor IL-I productionThe cell concentration was adjusted to 2 x 106 cells/ml incomplete medium, i.e. RPMI 1640 (GIBCO, Paisley, UK)supplemented with 5% fetal calf serum (FCS; National Veterin-ary Institute), 2 mM L-glutamine, 50 mm 2-mercaptoethanol, and50 mg/ml gentamycin (Sigma, St Louis, MO). Tissue cultureplates (Flow Labs, Irvine, UK) containing 0-2 ml/well of cellsuspension were incubated for 3 days at 37 C in 7% CO2 toallow the spontaneous release of IL-1 [24]. Thereafter, themedium was removed and the cells were washed with freshmedium. Antigen was added to triplicate wells at a finalconcentration of 0-2 pg protein/ml in complete medium. Matrixwas used at a concentration of 0-2 pg cholesterol/ml. Additionalwells received complete medium only, and were used asbackground controls. The plates were incubated again and ateach time point (as shown in Fig. 1) the supernatants werecollected for analysis of sIL-1, and the cells were treated forfurther detection of mIL-I .

Detection ofmIL-I and sIL-I by bioassayCultured cells were washed with medium, p-formaldehyde fixedand incubated overnight at 37°C to allow the release of sIL- Ipossibly present at the cell membrane [24]. The mIL-I wasmeasured as described [24]. Briefly, Dl0.G4.1 cells (D110) werepropagated in the presence of 10% supernatant ofconcanavalinA (Con A)-stimulated SC [25]. The D1O cells were harvested,washed and distributed at a concentration of 3 x 104 cells/wellcontaining the p-formaldehyde-fixed SC in a total volume of200pl. The plates were incubated for 72 h at 37°C, in the presence of2 pCi/well 3H-thymidine (5 Ci/mmol; Amersham, UK) duringthe last 4 h. Supernatant from LPS-treated SC was used aspositive control for interassay variability, and medium alone asnegative control for the proliferation of D1O cells. 3H-thymidineincorporation was measured in a Betaplate counter (Pharmacia,Uppsala, Sweden). The proliferation ofD10 cells is expressed asct/min.

For the detection of sIL- I by bioassay, 3 x 104DO cells wereadded to 50-pl aliquots of supernatants from cultured SC, and

(a) mlL-I

200 L A-

100

0 10 20 30

(c)

in200-

x

'E 100

0

0 0 0 30(e)

(b) slL-I50

25

0 10 20 30 40

(d)100

50

0 10 20 30 40

0 10 20 30 0 10 20 30 40Hours of incubation

Fig. 1. Kinetics of membrane-associated IL-I (mIL-l) and soluble IL- 1(sIL-1) in splenocytes from non-immunized or immunized mice meas-ured in a bioassay after stimulation in vitro with: 0, immuno-stimulatingcomplexes (iscoms) containing influenza virus envelope proteins; *,matrix of iscoms; or 0, micelles containing influenza virus envelopeproteins. (a, b) Splenocytes from non-immunized mice. (c, d) Spleno-cytes from iscom-immunized mice. (e, f) Splenocytes from micelle-immunized animals. Background values ranged between 0 1 and 2 x 103ct/min for all groups, except (e), which was 31 x 103 ct/min.

the total volume was adjusted to 200 pl/well. Thereafter theassay was run as described above for mIL-1.

ELISA for detection ofsoluble IL-IAliquots of 100 p1 of cell supernatants were tested in ELISA.Briefly, EIA/RIA plates (Costar, Cambridge, MA) were coatedwith 3 ,ug/ml of a MoAb hamster anti-mouse IL- 1 a in 50 mMcarbonate buffer pH 9-6. Empty sites were blocked with 5%skim milk sol in PBS. Duplicate samples were incubatedovernight at 4°C. Bound IL-la was detected using a biotin-labelled rabbit anti-mouse IL-ia and streptavidin-peroxidaseconjugate. Bound complexes were detected by reaction withtetramethyl-benzidine (TMB) and H202. Absorbance was readat 450 nm. The concentration of IL-ha in the samples wascalculated as pg/ml using a recombinant mouse IL-ha as astandard. Antibody and recombinant IL-I were purchased fromGenzyme (Cambridge, MA).

Inhibition of DJO cells proliferation by antibodiesThe antibodies used were: goat anti-murine IL-1 50 pg/ml(Advanced Magnetics Inc, Cambridge, MA), rat anti-murineIL-2 MoAb (50 pg/mI), 50pl supernatant from a rat anti-murineIL-4 MoAb IBlI 1, rat anti-murine IL-6 MoAb (2 pg/ml),rabbit anti-mouse tumour necrosis factor-alpha (TNF-a) 50 pg/ml, hamster anti-mouse IFN-Y MoAb (3 pg/ml). Antibodiesreacting with IL-2, IL-6, TNF-a and IFN-y were dilutedaccording to the instructions supplied by the manufacturer(Genzyme, Boston, MA). SC supernatants (50 pi) from non-primed mice stimulated with iscoms for 24 h were incubated

121

M. Villacres-Eriksson et al.

with 50 y1 anti-cytokine antibody for 30 min at 370C. Thesupernatant-antisera mixtures were added to Dl0 cell culturesand the proliferation was measured as described. Controlexperiments showed that these amounts of antibodies wereinhibitory for the respective recombinant cytokine (all cytokinesfrom Genzyme).

Preparation of adherent and non-adherent cell culturesSC from non-primed mice were incubated for 3 days, then thenon-adherent cells were transferred to a new plate. The cellsremaining in the wells (adherent) and the transferred cells (non-adherent) were stimulated with 0-2 yg/ml iscoms or matrix.Additional wells received medium alone and were used asbackground controls. Supernatants were collected after 24 hincubation at 370C and tested for IL-1 bioactivity.

Double immunofluorescence stainingSC (2 x 106/ml) were cultured with iscoms or matrix (0-2 yg/ml)or medium only for 16 h (non-primed mice) or 6 h (iscom-primed mice). The incubation times were selected according tothe bioassay results. The non-adherent cells were washed withPBS and stained for mIL- I a by incubation with goat anti-mouseIL-I a and donkey F(ab')2 anti-goat fluorescein conjugate(Jackson ImmunoResearch, West Grove, PA). Thereafter thesamples were stained for surface immunoglobulin by incubationwith a goat F(ab')2 anti-mouse immunoglobulin rhodamineconjugate (Jackson ImmunoResearch). The stained cells werefixed in suspension with 1-8% p-formaldehyde in PBS. Thesamples were then dried-mounted and inspected in a Nikonmicroscope fitted with blue and red filters. Cells showing greenfluorescence were scored as IL-1 + and those displaying orange-red fluorescence were considered to be immunoglobulin-posi-tive. Controls using a normal goat serum instead of anti-IL-iantibody did not show green fluorescence.

Table 1. The concentration of IL-la in supernatants from spleno-cytes cultured with immuno-stimulating complexes (iscoms),

micelles or matrix, as measured in ELISA*

IL- 1 a in supernatants (pg/ml)

In vitro stimulation witht

iscoms micelle matrix medium

A Non-primed mice 28 (7)4 4 (1) 18 (3) 3 (1)B Iscom-primed mice 3 (04) 0 13 (3) 0

Micelle-primedmice 20 (3) 0 22 (6) 0

* Cells cultured with 25 yg/ml lipopolysaccharide (LPS) pro-duced 11-5 pg/ml IL-la.

t Twenty-four hour supernatants from the cultures shown inFig. 1.

I S.D. are in parentheses.

1001-

o

Ec:3

Statistical analysisA paired-sample t-test was used for data evaluation.

RESULTS

Kinetics of the expression of IL-IIn cells from non-primed mice, iscoms, matrix or micellesinduced production ofmIL- I, with maximal amount detected atabout 16 h (Fig. Ia). Matrix induced about three times moremIL-I (114000 ct/min) than iscoms did. Micelles induced twiceas much mIL- I as matrix. The production of sIL- 1 induced bymatrix and iscoms peaked at 24 h. Matrix stimulated five timesas much sIL- I as iscoms. Micelles did not induce sIL- I (Fig. I b).

Splenocytes from iscom-immunized mice incubated for 6 hwith matrix responded with significantly higher production ofIL-I (41 000 ct/min) than cultures incubated with iscoms ormicelles (Fig. Ic). The production of sIL-I peaked at 24 h.Matrix induced about six times more sIL-I than iscoms.Micelles did not induce production of sIL-1 (Fig. Id).

Splenocytes from micelle-immunized mice stimulated withiscoms or matrix induced high levels of mIL-I at 6 h (152000and 187000 ct/min respectively). Micelles induced a delayed,protracted and low response (36000 ct/min at 24 h, Fig. le).Soluble IL-I was maximal at 24 h and matrix induced about500/) more sIL-I than iscoms did. Micelles did not stimulateproduction of sIL- I over the background (Fig. If).

501-

01

7

|___

-_K I

Total Adherent Non-adherentpopulation cells cells

Fig. 2. Adherent cells produce soluble IL-1 (sIL-l) as measured bybioassay. The proliferation of DIO cells induced by IL-1 in thesupernatants from spleen cell cultures was measured by 3H-thymidineuptake. Spleen cells were stimulated with immuno-stimulating com-plexes (iscoms) (U) or matrix (0).

Detection ofsIL-J in ELISAReplicates of the supernatants tested in bioassay were tested inan ELISA specific for IL-lIa. Values ranging between 13 and 29pg/ml IL-la were obtained in supernatants from splenocytesfrom non-primed mice or micelle-primed mice stimulated invitro with iscoms or matrix (Table 1). Low values were detectedin supernatants from splenocytes from iscom-primed micecultured with iscoms (3 pg/ml). Supernatants from cells culturedwith micelles contained low concentrations of IL-la in allgroups.

Secretion of IL-1 by splenic adherent cellsCulture supernatants from splenic adherent cells stimulatedwith iscoms or matrix contained sIL- 1 (25 000 and 53 000 ct/minrespectively, Fig. 2). These values were 36% and 47% respect-ively of the values induced by supernatants from cultures

L-L ;

122

Induction of IL-I by iscoms

Anti-IL-la

Anti-IL-2

Anti-I L-4

Anti-I L-6Anti-TNF-a

Anti-IFN-y

0 50 100Per cent of inhibition

Fig. 3. Antibody to IL-Ia inhibits the proliferation of D10 cells inducedby supernatants from splenocytes stimulated with immuno-stimulatingcomplexes (iscoms) in vitro. The following antibodies were tested: IL-I a(0), IL-2 (U), IL-4 (U), IL-6 (U), tumour necrosis factor-alpha (TNF-a)(U), or IFN-y (U).

Table 2. The proportion of immunoglobulin- and mIL-i-producingnon-adherent cells in splenocyte cultures from non-primed and primedmice stimulated in vitro with immuno-stimulating complex (iscom) or

matrix

Percentage cells stained for

Per cent IL-1 + ofImmunoglobulin+ IL-I + immunoglobulin+

Non-primed mice* stimulated:with medium 45 9 (2-9)t 1 9 (0) 3-3 (0-4)with matrix 46 8 (3 2) 2-6 (0-2) 5 5 (0 6)with iscom 44 9 (0 6) 2 6 (0 4) 5 4 (1 4)

Iscom-immunized micet stimulated:with medium 45-8 (2 3) 3-1 (0-5) 5-6 (0 5)with matrix 44-8 (3 7) 2 8 (0 2) 5 0 (0-2)with iscom 45-3 (4-3) 3-2 (0 2) 7-0 (0-2)

* Cells from non-primed mice (n= 3) were treated in vitro only.t S.D. are in parentheses.t Mice (n =3) were immunized with influenza iscoms. Four days

later the spleen cells were collected and stimulated in vitro.

containing both adherent and non-adherent populations. Nostimulation was recorded in wells containing supernatants fromnon-adherent cells cultured with iscoms or matrix.

Inhibition ofIL-l-mediated proliferation ofDJO cells by anti-IL-la antibodiesThe antibodies reacting with IL-la blocked the stimulation ofproliferation of DIO cells by 79%, i.e. the 3H-thymidine uptakewas 67 000 ct/min without antibody and 14 000 ct/min with anti-IL-la antibody. None of the other antibodies inhibited thestimulation of DIO cells (Fig. 3).

The effect ofstimulation with iscoms or matrix on the expressionofmIL-J by B cellsIt is well established in the literature that adherent cells produceIL-i [4]. Here we analyse if splenic non-adherent cells producemIL-1 upon iscoms or matrix stimulation. The depletionexperiments described above showed that non-adherent spleno-cytes did not secrete the cytokine. The presence of mIL-I on

splenic B cells from non-primed or iscom-primed mice was

studied by double immunofluorescence (Table 2). In general, thetotal population of non-adherent cells expressing mIL-1 was

around 3% (1 9-3-2%). The number of immunoglobulin-positive B cells expressing mIL-l ranged from 3-3% to 7-0%.The lowest value (3 3%) was found for spleen cells from non-

treated mice cultured with medium only. Otherwise the rangewas between 5 4% and 7T0%. No significant difference was seen

in this respect between spleen cells obtained from non-primed or

iscom-primed mice.

DISCUSSION

For some substances with adjuvant activity such as LPS, MDP,cholera toxin, Freund's adjuvants, IL-I seems to be an impor-tant mediator [6,7]. Although multimeric forms of an antigenare considerably more immunogenic than the monomeric formof the same antigen [8,9], the capacity of multimers to enhancethe immune response has not been related with the productionof cytokines, except for Listeria bacterial antigen. However,bacteria contain LPS and toxins which may act as mediators forcytokines [4]. Monomeric forms of proteins like BSA, which arepoorly immunogenic, do not induce mIL-1 or sIL-l [4]. In thisstudy we demonstrated that the envelope proteins of influenzavirus in the micelle form and in the absence of an adjuvantinduce production of mIL-I but not sIL-I in vitro.

It is noteworthy that matrix, and to a lesser extent iscoms,induced the production of mIL-1 and sIL-1, as shown inbioassay and confirmed by immunoassay. The immunoassay, incontrast to the bioassay, showed that iscoms induced produc-tion of sIL-I by cells from non-primed mice. It is possible thatother factor(s) present in these supernatants down-regulate theIL-1 receptor in the responding DlO cells, or alternatively, theIL-I produced is not bioactive. In all groups, matrix inducedhigher production of mIL-I and sIL-I than iscoms, probablydue to the fact that the matrix contained twice as much of theQuil A components (data not shown). Micelles did not induceproduction of mIL- I in SC from mice primed 4 days previouslywith iscoms. The production of mIL- I induced by micelles wasrestricted to SC from naive mice or from mice primed withmicelles. SC from mice primed with iscoms 4 days earlierproduced low levels of mIL-I or sIL-1 in response to in vitrotreatment with iscoms. These results are in agreement with thoseof Fossum et al., which showed that the spontaneous prolifera-tion ofSC from iscom-primed mice was depressed 3 days but not7 or 11 days after the last treatment with iscoms [20]. Oneexplanation is that iscoms, but not micelles, induce productionof IFN-y by SC of primed mice [19,21], which is known todecrease the IL-l-induced IL-I release [26]. Generally, mIL-Iwas more readily induced than sIL- 1, and reached peak values at3-6 h, while sIL-I was detected between 12 h and 30 h. Overall,the mIL-I values were higher than those of sIL-I.

Although only the adherent SC were able to respond withsIL-I production, they seemed to benefit from factor(s) pro-

123

124 M. Villacres-Eriksson et al.

vided by the non-adherent cells, since the production of sIL-lwas increased in the presence of non-adherent SC. Highlypurified splenic B or T cells do not produce sIL-1 in response tostimulation with iscoms, while peritoneal cells produce IL-1upon iscom treatment in vivo (manuscript in preparation). Theproduction of IL-1 in LPS or cholera toxin-stimulated murinecells is mediated by macrophages [7,27], and the production ofIL-1 by peritoneal macrophages, cultured with heat-killedbacteria as antigen, was enhanced by addition ofT cell products[28].

The capacity ofiscoms and matrix to induce mIL- 1 in B cells,which are important APC (reviewed in [29]) was also tested, butthese preparations induced low increases in the percentage ofmIL-i + non-adherent cells. A small proportion (<1%) of thecells was s~g- but mIL-i +, and probably contained polymor-phonuclear cells, natural killer cells or dendritic cells, all ofwhich can produce IL- 1 [3]. The frequency of these cells was thesame in primed or non-primed SC, cultured with medium onlyor stimulated with iscoms or matrix (data not shown).

IL-I is important for initiation of the immune response, e.g.by increasing the capacity of macrophages to present antigen toCD4+ T cells, which is to a great extent dependent on thepresence of Ta molecules on the presenting cell [30]. Liposomesbearing antigen and Ta molecules were more efficient as syntheticAPC when mIL- I was also included [31]. In experiments cross-linking the T cell receptor (TCR), purified CD4 or CD8 T cellsrequired addition of IL-I or IL-6 to proliferate or secrete IL-2[32]. IL-1 and GM-CSF are considered important to increasethe capacity of dendritic cells to present antigen to T cells in theprimary immune response [33]. Resting or LPS-activated B cellshave increased capacity to present antigen to primed T cellsupon addition of IL- I or IL-2 [34]. Two reports have shown thatiscoms induce increased expression of Ta in an antigen-specificmanner both in vivo and in vitro [18,19]. In conclusion, theinduction of IL-I by iscoms is likely to be important for theinitiation of the immune response, and probably plays asignificant role in the high immunogenicity in vivo of iscom-borne antigen.

ACKNOWLEDGMENTSWe thank Professor Hans Wigzell for the gift of 1 IBl 1 supernatant,Professor Gunnar Alm and Dr Howard Etlinger for critical reading ofthe manuscript. This study was financially supported by the SwedishMedical Research Council and the Swedish Cancer Society.

REFERENCES1 Unanue ER, Allen PM. The basis of the immunoregulatory role ofmacrophages and other accessory cells. Science 1987; 236:551-7.

2 Dinarello CA. Interleukin-l. Rev Infect Dis 1984; 6:51-95.3 Nathan CF. Secretory products of macrophages. J Clin Invest 1987;

79:319-26.4 Kurt-Jones EA, Virgin H, Unanue ER. In vivo and in vitro

expression of macrophage membrane interleukin 1 in response tosoluble and particulate stimuli. J Immunol 1986; 137:10-14.

5 Dinarello CA. The biology of interleukin-l. Chem Immunol 1992;51: 1-32.

6 Oppenheim JJ, Togawa A, Chedid L, Mizel S. Components ofmycobacteria and muramyl dipeptide with adjuvant activity inducelymphocyte activating factor. Cell Immunol 1980; 50:71-81.

7 Bromander A, Holmgren J, Lycke N. Cholera toxin stimulates IL-Iproduction and enhances antigen presentation by macrophages invitro. J Immunol 1991; 146:2908-14.

8 Morein B, Helenius A, Simons R et al. Effective subunit vaccinesagainst an enveloped animal virus. Nature 1978; 276:715-8.

9 Morein B, Simons K. Subunit vaccines against enveloped viruses:virosomes, micelles and other protein complexes. Vaccine 1985;3:83-93.

10 Morein B, Ekstr6m J, Lovgren K. Increased immunogenicity of anon-amphipathic protein (BSA) after inclusion into iscoms. JImmunol Meth 1990; 128:177-81.

11 Lbvgren K, Lindmark J, Pipkorn R, Morein B. Antigenic presen-tation of small molecules and peptides conjugated to a preformediscom as carrier. J Immunol Meth 1987; 98:137-43.

12 Morein B, L6vgren K, H6glund S, Sundquist B. The iscom: animmunomodulating complex. Immunol Today 1987; 8:333-8.

13 Takahashi H, Takeshita T, Morein B et al. Induction of specificCD8+ class I-MHC restricted CTL against HIV envelope protein byimmunization with purified whole protein in iscoms. Nature 1990;344:873-5.

14 Jones PD, Tha Hla R, Morein B, Ada GL. Cellular immuneresponses in the murine lung to local immunization with influenza Avirus glycoproteins in micelles and immunostimulatory complexes(iscoms). Scand J Immunol 1988; 27:645-52.

15 Heeg K, Kuon W, Wagner H. Vaccination of class I majorhistocompatibility complex (MHC)-restricted murine CD8+ cyto-toxic T lymphocytes towards soluble antigens: immunostimulating-ovalbumin complexes enter the class I MHC-restricted antigenpathway and allow sensitization against the immunodominantpeptide. Eur J Immunol 1991; 21:1523-7.

16 Mowat A, Donachie AM. Iscoms-a novel strategy for mucosalimmunization? Immunol Today 1991; 12:383-5.

17 Watson DL, Lovgren K, Watson NA et al. Inflammatory responseand antigen localization following immunization with influenzavirus iscoms. Inflammation 1989; 13:641-9.

18 Watson DL, Watson NA, Fossum C et al. Interactions betweenimmune-stimulating complexes (ISCOMs) and peritoneal mono-nuclear leucocytes. Microbiol Immunol 1992; 36:199-203.

19 Bergstr6m-Mollaoglu M, L6vgren K, Akerblom L et al. Antigen-specific increases in the number ofsplenocytes expressing MHC classII molecules following restimulation with antigen in various physicalforms. Scand J Immunol 1992; 36:565-74.

20 Fossum C, Bergstrom M, Lovgren K et al. Effect of iscom and/ortheir adjuvant moiety (matrix) on the initial proliferative and IL-2responses in vitro. Comparison of spleen cells from mice inoculatedwith iscoms and/or matrix. Cell Immunol 1990; 129:414-25.

21 Villacres-Eriksson M, Bergstrom-Mollaoglu M, Kaberg H, MoreinB. Involvement of interleukin-2 and interferon-gamma in theimmune response induced by influenza virus iscoms. Scand JImmunol 1992; 36:421-6.

22 Sundquist B, L6vgren K, Hoglund S, Morein B. Influenza virusiscoms: biochemical characterization. Vaccine 1988; 6:44-48.

23 Lovgren K, Morein B. The requirement of lipids for the formation ofimmunostimulating complexes (iscoms). Biotechnol Appl Biochem1988; 10:161-72.

24 Kurt-Jones EA, Beller DA, Mizel SB et al. Identification of amembrane-associated interleukin- 1 in macrophages. Proc NatlAcad Sci USA 1985; 82:1204-8.

25 Helle M, Boeije L, Aarden LA. Functional discrimination betweeninterleukin 6 and interleukin 1. Eur J Immunol 1988; 18:1535-40.

26 Ghezzi P. Dinarello CA. IL- I induces IL- 1. III. Specific inhibition ofIL-1 production by IFN-y. J Immunol 1988; 140:4238-44.

27 Koide S, Steinman R. Induction of murine IL-1: stimuli andresponsive primary cells. Proc Natl Acad Sci USA 1987; 84:3802-6.

28 Weaver CT, Duncan LM, Unanue ER. T cell induction ofmacrophage interleukin-1 during antigen presentation. J Immunol1989; 142:3469-76.

29 Lanzaveccia A. Receptor-mediated antigen uptake and its effects onantigen presentation to class II restricted T lymphocytes. Ann RevImmunol 1990; 8:773-93.

Induction of IL-I by iscoms 125

30 Kurt-Jones EA, Virgin HW, Unanue ER. Relationship of macro-phage Ia and membrane IL- I expression to antigen presentation. JImmunol 1985; 135:3652-4.

31 Bakouche 0, Lachman LB. Antigen presentation by liposomesbearing class II MHC and membrane IL-1. Yale J Biol Med 1990;63:95-107.

32 Stein PH, Singer A. Similar costimulation requirements of CD41and CD8 I primary T helper cells: role of IL- I and IL-6 in inducingIL-2 secretion and subsequent proliferation. Int Immunol 1992;4:327-35.

33 Naito K, Inaba K, Hirayama Y et al. Macrophage factors whichenhance the mixed leukocyte reaction initiated by dendritic cells. JImmunol 1989; 142:1834-9.

34 Frohman M, Cowing C. Presentation of antigen by B cells:functional dependence on radiation dose, interleukins, cellularactivation, and differential glycosylation. J Immunol 1985;134:2269-75.