Modes of action of Freund’s adjuvants
in experimental models of autoimmune diseases
Alfons Billiau and Patrick Matthys
Rega Institute, University of Leuven, Belgium
Abstract: Freund’s adjuvants are irreplaceable
components of induction protocols of many exper-
imental animal models of autoimmune disease.
Apart from the early studies done in the 1950s and
1960s, no further direct investigation on the mode
of action of these adjuvants has been undertaken.
It is generally assumed that incomplete (IFA) and
complete Freund’s adjuvant (CFA) act by prolong-
ing the lifetime of injected autoantigen, by stimu-
lating its effective delivery to the immune system
and by providing a complex set of signals to the
innate compartment of the immune system, result-
ing in altered leukocyte proliferation and differen-
tiation. Here, we review evidence collected from
various types of studies that provide more insight in
the specific alterations of the immune response
caused by IFA and CFA. Early events include rapid
uptake of adjuvant components by dendritic cells,
enhanced phagocytosis, secretion of cytokines by
mononuclear phagocytes, and transient activation
and proliferation of CD4ⴙ lymphocytes. The my-
cobacterial components within CFA signal T lym-
phocytes to assume a Th1 profile so that strong
delayed-type hypersensitivity against autoantigens
develops. In the absence of mycobacteria, T-lym-
phocyte differentiation tends to assume a Th2 pro-
file with strong antibody production only. The myco-
bacterial component also accounts for a morphologic
and functional remodeling of the haemopoietic system
that develops over a period of several weeks and that
is characterized by a drastic expansion of Mac-1ⴙ
immature myeloid cells. These cells have been
found to be associated with enhanced disease in
some models but with reduced disease in others.
Thus, in experimental autoimmune diseases, CFA-
mediated activation of the innate immune compart-
ment is important not only by regulating the early
induction phase but also by providing a surplus of
effector and regulator cells in the late phase.
J. Leukoc. Biol. 70: 849 – 860; 2001.
Key Words: Freund 䡠 adjuvant 䡠 dendritic cells
INTRODUCTION
For half a century, incomplete (IFA) and complete Freund’s
adjuvant (CFA) have been the most commonly used immuno-
adjuvants for experimental work. Nevertheless, their mode of
action is still not completely understood. More so in the face of
enormous progress in the knowledge of the molecular and
cellular basis of the immune system, immunologists have con-
tinued to use CFA without really caring about its mode of
action. Invariably, the use of CFA in experimental protocols is
hidden in the Materials and Methods sections of experimental
studies without further consideration of the implications in the
Discussion sections. The use of CFA is mandatory in the
induction protocols of many experimental models of autoim-
mune disease, such as experimental autoimmune encephalo-
myelitis (EAE), neuritis (EAN), uveitis (EAU), thyroiditis
(EAT), and orchitis. Here, we review older and more recent
studies pertaining to the molecular and cellular basis of the
mode of action of CFA in these models. Autoimmune diseases
in animal models involve components of innate and acquired,
as well as cellular and humoral, immunity, and a central
question in considering the role of CFA is whether it merely
boosts the autoantigen-specific response or whether its effect as
a stimulant of innate immunity is in itself also important.
BRIEF HISTORY
IFA is essentially paraffin oil containing mannide mono-oleate
as a surfactant (for a detailed description, see ref. [1]). When
mixed with aqueous solutions or suspensions of antigens, the
adjuvant forms a viscous water-in-oil emulsion, with the anti-
gens in the water phase. In addition, CFA contains heat-killed
mycobacteria (Mycobacterium tuberculosis or other) and is more
potent and more adequate for certain purposes. Two lines of
investigation are at the basis of the decades-long success of
CFA as an instrument in the hands of immunologists. Injec-
tions of mycobacteria in a fatty excipient were first used by
Rabinovitch in Koch’s laboratory in an attempt to understand
why pathogenic mycobacterium species did, and nonpatho-
genic ones did not, induce formation of tubercle lesions. In a
historical account of 1956 [1], Freund recounts Rabinovich’s
finding: “. . . that Mycobacterium butyricum injected in exper-
imental animals caused lesions at the sites of injection some-
what resembling tubercles, but when the bacteria were incor-
Correspondence: A. Billiau, Rega Institute, University of Leuven, Labora-
tory of Immunobiology, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
E-mail: Alfons.Billiau@Rega.Kuleuven.ac.be
Received February 26, 2001; revised August 30, 2001; accepted September
13, 2001.
Journal of Leukocyte Biology Volume 70, December 2001 849
porated into butter, they evoked a cellular reaction almost
identical with a tubercle caused by pathogenic tubercle ba-
cilli. . . ,” and further, “Aiming to show that butter had no
specific role in this respect, Grasberger reported that paraffin
oil was far more effective.”
A second line of investigation began with the observation
that guinea pigs infected with M. tuberculosis respond differ-
ently to immunization with unrelated antigens, e.g., sheep red
blood cells (SRBC). If injected together with a subsequent
inoculation of the mycobacteria, production of antibodies to
SRBC was increased [2]. More importantly, the dermal hyper-
sensitivity response was of a delayed rather than an immediate
allergic type, as was the case in noninfected animals. This
observation was made by Dienes [3], who wanted to sensitize
guinea pigs against “protein substances” in a way that would
result in skin reactivity with the characteristics of the “tuber-
culin sensitiveness” (i.e., time course, characteristic superfi-
cial appearance, absence of connection with serum antibody,
failure of passive transfer). He found that this could be
achieved when the protein antigen (egg white, horse serum,
timothy pollen, etc.) was injected in tuberculous lesions of
infected animals.
Freund was intrigued by this phenomenon. In particular, he
wanted to see whether the delayed-type hypersensitivity (DTH)
sensitization, obtained by exposure to mycobacteria, could be
transferred from one animal to another by lymph node cells.
For this purpose, it was desirable to avoid working with in-
fected donors. Hence, he tried to repeat Dienes’ work using
killed mycobacteria instead of a plain infection. Not being
successful right away, he started using suspensions of myco-
bacteria in paraffin oil, as had been done by those working on
pathogenesis of the tubercle lesion. Serendipity apparently
came in when, being unsuccessful in attaining his primary aim,
he wanted to recycle sera of redundant guinea pigs as a source
of complement. The sera of these animals, which had received
mycobacteria in oil, gave apparently “false-positive” comple-
ment-fixation reactions with mycobacteria. An analysis of these
results revealed that the animals, although having received
only a single antigen exposure, had developed high antibody
titers to mycobacterial antigens; i.e., adding the oil to the
heat-killed mycobacteria had resulted in an unprecedented
stimulatory effect on immunization.
This was the beginning of the use of CFA for the purpose of
producing potent antisera against soluble antigens. Its use as
an instrument to induce experimental autoimmune disease
dates back to the late 1940s and early 1950s, when models
such as EAE, EAN, EAU, and allergic aspermatogenesis were
first described. Already in 1933, Rivers et al. [4] (loc. cit. ref.
[1]) had described induction of encephalomyelitis in monkeys.
Following repeated injections of brain autolysates without any
adjuvant, some monkeys developed what is now known as
EAE. However, in the late 1940s, introduction of the use of
CFA allowed induction of the disease with a single injection of
brain material in several animal species, e.g., in guinea pigs
[5]. Variants of this procedure are still used today for induction
of EAE. In addition, however, experimental models for other
organ-specific autoimmune diseases most often, although not
obligately, rely on the use of CFA, i.e., EAU, EAN, experi-
850 Journal of Leukocyte Biology Volume 70, December 2001
mental autoimmune orchitis, experimental autoimmune thy-
roiditis, CIA, and myasthenia gravis.
ACTIONS OF CFA
Adjuvancity: hyperimmunization and facilitation
of autoimmune disease induction
A prototype scheme for the use of CFA to hyperimmunize
experimental animals consists of administering a CFA-emul-
sified antigen at one or several subcutaneous sites, followed by
one or several booster injections, each given with intervals of
weeks or months, mostly in plain aqueous medium or as an
emulsion in IFA. The procedure results in high levels of
circulating antigen-specific antibodies, in strong ex vivo T-
lymphocyte responsiveness, and in DTH in vivo. Experimental
protocols for induction of autoimmune disease vary consider-
ably depending on the target organ concerned and on the
animal species or strain. In fact, the use of CFA is not always
an absolute requirement. For instance, in certain strains of
rats, EAE can be induced without CFA, whereas in guinea pigs
and in mice, CFA is mandatory (for review, see ref. [6]). Some
induction schedules for EAE even include the Bordetella per-
tussis vaccine for optimal reproducibility. Even in highly sus-
ceptible mouse strains such as SJL/J or Biozzi ABH mice,
immunization schedules without any adjuvant, or with IFA or
other adjuvants, fail to induce disease symptoms. For induction
of autoimmune arthritis, CFA is less crucial. Immunization of
rats with collagen-II in IFA results in a form of protracted
polyarthritis called “collagen-induced arthritis” (CIA) [7]. The
same is true for inducing the disease in DBA/1 mic; however,
with CFA instead of IFA, the incidence and severity are much
higher [8, 9].
In certain strains of rats, treatment with IFA or CFA, without
any joint-specific antigen, can cause arthritis [10]. Arthritis
induced by IFA is called oil-induced arthritis (OIA); it is an
acute and self-limited affection [11]. Other oils such as
pristane [12] and squalene [13], the latter being a normal body
component, have also been found to induce arthritis in sus-
ceptible rats or mice [14].
Adjuvant-induced arthritis (AIA), inducible by CFA (with-
out added autoantigen) in rats, is a chronic disease [15]. In
Lewis rats, it develops in two phases: an acute periarticular
inflammation followed by a phase of bone involvement [16].
The investigators isolated arthritogenic T cell clones, which
recognize epitopes of the mycobacterial heat shock protein
(HSP), hsp65. This has led to the assumption that the patho-
genesis is closely linked to the immune response to these
mycobacterial antigens. Antibodies and specific T cells against
hsp65 epitopes do cross-react with epitopes on host HSP. The
pathogeneses of oil-induced and adjuvant-induced arthritis are
closely related. Both diseases are CD4⫹ T-cell-dependent [14,
17], and both are associated with an immune response to HSP.
However, the overall in vivo effect of the anti-HSP response is
protective rather than disease-promoting [18]. In addition,
injection of IFA can prevent induction by CFA [12].
In MRL-lpr mice, which spontaneously develop an arthritis-
like disease, CFA was found to accelerate and to increase the
http://www.jleukbio.org
incidence of arthritis [19]. It is interesting that adjuvant-
injected mice showed enhanced circulating antibodies against
collagen types I and II and DNA.
Local inflammation
In view of the numerous published studies in which CFA has
been used in the second half of the 20th century, it is hard to
imagine that immunologists would have reached their current
state of knowledge without having had access to this powerful
tool. Nevertheless, the procedure today is increasingly being
subjected to institutional and governmental regulatory restric-
tions [20 –22]. The reason is that within days after the subcu-
taneous administration of CFA, a strong and long-lasting in-
flammatory reaction appears at the site of injection and in the
draining lymph nodes. In some cases, this inflammation may be
excessively painful to the animal, depending on the evolution
in time and on the injection site. Often the lesions evolve to
become ulcera. In rabbits, rats, and mice, the footpad is an
injection site reputed not only for its effectiveness in terms of
producing a strong immune response, but also for its obvious
painfulness to the animal.
Granuloma formation
Concomitantly with inflammation at the injection site, hyper-
plasia and architectural changes take place in regional and
distant lymph nodes. In early studies, repeated injection of
killed mycobacteria incorporated in paraffin oil has been seen
to induce tubercle-like lesions in lymph nodes but also in
nonlymphoid tissues [1, 23, 24]. Local and distant granulomas
that form following local injection of IFA are mainly composed
of mononuclear phagocytes (MPCs) and have the appearance of
a foreign-body tissue reaction with oil drops in between cells
and in the phagocytes. When CFA is used, the granuloma
formation is more vigorous. Also, the phagocytes can take an
epitheloid appearance and can contain acid-fast rods. Occa-
sionally, typical tubercles with Langhans giant cells do occur
[1, 25]. Clearly, the host response to CFA should be seen as
resembling one coping with a slowly fading primary infection
with living mycobacteria.
Induction of cells with suppressor activity
Splenocytes of guinea pigs immunized with ovalbumin in CFA
were shown to suppress the in vitro mitotic response of lymph
node cells from ovalbumin-sensitized indicator guinea pigs to
the antigen or to concanavalin A (Con A) [26]. This suppressor
activity was associated with a nonadherent effector cell popu-
lation. Although there appears to have been no explicit fol-
low-up of this observation, infection of mice with Mycobacte-
rium lepraemurium has been shown to result in similar gener-
ation of suppressor splenocytes, demonstrable by an inhibitor
effect on the expression of DTH reactivity in indicator animals
[27, 28] or on the mitotic response of indicator lymphocytes to
Con A [29, 30]. Again, the effector cells appeared to be
nonadherent and radioresistant. In fact, they were found to
evolve in three stages: 1) generation of radiosensitive precur-
sors in the infected mouse, 2) maturation upon overnight in
vitro culture, and 3) activation following exposure to the Con
Billiau and Matthys Freund’s adjuvants in autoimmune diseases
A-stimulated indicator splenocytes. Stage 1, in vivo, was found
to depend on activity of sensitized lymphocytes; stage 2 re-
quired the presence of a nonadherent, non-T, non-B, non-
natural killer (NK) cell population; and stage 3 was distin-
guishable from the preceding stage by its being dependent of
the presence of interferon-␥ (IFN-␥).
Protection against fetal loss
In a mouse model of fetal resorption as a result of genetic
disparity between the pregnant female and the mating male
(CBA/J females mated to DBA/2J males), injection of CFA
exerts a protective effect against fetal loss [31]. The underlying
mechanisms have not been clarified. Significantly, however,
the protective effect is transferrable with splenocytes from
CFA-prepared nonpregnant females. It is also associated with
increased invasion of Mac-1⫹ cells in the placenta, reduced ex
vivo production of IL-2 by splenocytes, decreased in vitro
embryotoxic effect of maternal serum [32], diminished re-
sponses of maternal splenocytes toward paternal allo-antigens,
suppressive effect of lymphocytes derived from spleen, lymph
nodes, or placenta for maternal lymphocyte responses to pa-
ternal antigens, and increased proportions of NK-like cells but
not of CD4⫹ or CD8⫹ cells in lymphoid organs [33].
Prevention of diabetes in nonobese diabetic
(NOD) mice
Treatment with only CFA has been shown to prevent develop-
ment of diabetes mellitus in NOD mice, whereas these mice
normally develop this autoimmune disease spontaneously [34].
Infection with live bacillus Calmette-Guerin (BCG) was seen to
exert a similar protective effect [35]. In both instances, a
“suppressor” Mac-1⫹ cell population was identified in the
spleens of treated mice. These cells could inhibit transfer of
disease by splenocytes of diabetic donors to unaffected recip-
ients and could also suppress T-cell responses of untreated
diabetic mice to mitogens or anti-CD3 antibody. Conversely,
BCG-infected NOD mice, although protected against diabetes,
do develop lupus-like disease [36].
THE ACTIVE COMPONENTS
The combination of paraffin oil and surfactant, the two compo-
nents of IFA, is in no way immunologically or pharmacologi-
cally inert. Aside from affecting trafficking of the embedded
antigens, IFA by itself exerts various effects on the immune
system, locoregionally and systemically. Thus, IFA stimulates
innate immunity, as evident by transiently increased resistance
to bacterial infection [37]. It also induces expression of cyto-
kines, predominantly tumor necrosis factor ␣ (TNF-␣) in re-
gional lymph nodes [38], and causes opening of the blood-brain
barrier [39]. The adjuvant effect of IFA on the immune re-
sponse to SRBC can be blocked by administration of antioxi-
dants, suggesting that IFA induces production of oxygen rad-
icals [40]. IFA can also trigger autoimmune-like disease, in
particular, arthritis, in genetically predisposed animals (vide
supra).
851
 An intensely investigated question is the chemical nature of
the mycobacterial substances that account for the immunoad-
juvant effects of CFA. This has led to identification of various
more or less complex molecules possessing adjuvant effects
similar to that of entire mycobacteria. One of these, muramyl
dipeptide (MDP), is a universally occurring building block of
the peptidoglycan component of the bacterial cell wall. For
certain purposes, MDP can replace the mycobacteria in CFA
but also possesses adjuvant effect without having to be incor-
porated in a water-oil emulsion. One aspect of the adjuvant
effect of these substances is their ability to stimulate haema-
topoiesis. For instance, adjuvant-active MDP was found to
induce a rise in the level of monocyte-macrophage colony-
stimulating activity in serum, expansion of granulocyte-mac-
rophage progenitors in the spleen, and proliferation of multi-
potential stem cells in bone marrow [41].
Glycolipids typical for Mycobacteria are trehalose dimyco-
late (TDM) and lipoarabinomannan. Mycolic acids are long-
chain branched ␤-hydroxy carboxylic acids that occur in My-
cobacteria, Nocardiae, and Corynebacteria. TDM is believed to
account for part of the adjuvant effects of CFA, one of the
arguments being that certain Nocardia and Corynebacterium
species can replace mycobacteria in CFA for induction of EAE
(for review, see ref. [6]). Also, TDM emulgated in a Tween/oil
excipient [42] was shown to sensitize mice against lipopoly-
saccharides (LPS).
Lipoarabinomannans (LAM) consist of a cell membrane-
bound lipid core, phosphatidylinositol, carrying a phosphodi-
ester-bonded heteropolysaccharide chain containing arabinose
and mannose residues. The chains cross the cell wall extending
to the external surface of the bacteria. In ManLAM, the poly-
saccharide chain terminates in a mannose cap, whereas in
AraLAM, the terminal suger is arabinose. Simpler versions are
LM (lipomannan), which lacks arabinose residues, and PIM
(phosphatidylinostol mannoside), which lacks arabinose and
most mannose residues. To some extent, all these versions are
biologically active; however, removal of the fatty acids from the
phosphatidylinositol core results in complete abrogation of
biological activity, indicating that this core is essential. Nev-
ertheless, the configuration of the polysaccharide side chain
can modulate biological activity [43]. Thus, the presence of the
mannose cap in ManLAM reduces the amplitude of the host
response, although it promotes uptake of the molecule into
MPCs [44]. LAM and PIM are agonists of the toll-like receptor
Tlr2 [45], and uptake of LAM by MPCs shares features with
uptake of LPS because the response to both is inhibited by
anti-CD14 antibodies [46]. Following uptake, LAM is traf-
ficked back to the cell surface in association with CD1 and
presented as a major histocompatibility complex (MHC)-inde-
pendent T-cell epitope. Cytokines and chemokines induced by
LAM in cultures of human peripheral blood mononuclear cells
(PBMC) are those typically produced by MPCs: interleukin
(IL)-1, TNF-␣, granulocyte-macrophage colony-stimulating
factor (GM-CSF), IL-6, IL-10, IL-8 [47], and monocyte che-
moattractant protein (MCP)-3 [48]. Genes shown to be induced
by LAM in murine macrophages are those of the chemokines
murine homologue of MCP-1 (JE) and KC and that of inducible
nitric oxide synthase (iNOS) [43]. Intravenous injection of
852 Journal of Leukocyte Biology Volume 70, December 2001
TDM in normal and preimmunized mice was shown to induce
pulmonary granulomas [49].
HSPs are other mycobacterial components that may play a
role in the biological effects of CFA. HSPs are intracellular
proteins occurring in prokaryotes as well as eukaryotes. Their
most important function is to act as chaperones for other
intracellular proteins during folding, unfolding, assembly, and
transport. Various conditions of cellular stress induce in-
creased production of HSPs, apparently as a mechanism to
safeguard the integrity of cellular proteins. However, in higher
eukaryotes, such increased production also regulates immune
responses [18]. HSPs can reach the extracellular fluid and bind
to cellular receptors. Human hsp60, in particular, binds to and
activates the toll-like receptor, Tlr4, resulting in an LPS-like
effect on mononuclear phagocytes [50, 51]. Thus, any stress
situation can provoke a “danger signal” resembling the one
given by bacterial LPS. Also, mycobacteria, although not pos-
sessing an LPS-containing outer cell wall, still can activate
mononuclear phagocytes through the same signaling cascade.
Another aspect of immunological activity of exogenous HSPs is
that the primary structure of this family of molecules has
remained highly conserved over evolution. Bacterial HSPs are
immunogenic in mammals, but the resulting immune response
cross-reacts with the human HSPs. This has evidently led to
speculation that HSPs of bacteria, which colonize or infect
higher animals, may act as the primary immunogens for initi-
ation of autoimmunity. However, clinical and experimental
evidence have indicated an opposite effect of HSPs [18].
Experimental immunization with HSPs leads mostly to a resis-
tance to subsequent induction of autoimmune disease. More-
over, in clinical settings, enhanced expression of endogenous
HSPs correlates with remission rather than exacerbation of
autoimmune disease parameters.
CpG oligodeoxynucleotides, present in the heat-killed my-
cobacteria, may participate in bringing about the biological
effects of CFA. DNA of bacteria, including mycobacteria,
differs from mammalian DNA by containing unmethylated CpG
dinucleotide motifs. CpG-containing oligonucleotides have
been shown to strongly stimulate innate immune mechanisms,
including production of cytokines by MPCs, maturation, and
activation of antigen-presenting cells (APCs) and Th1 skewing
of the immune response in vitro and in vivo (for review, see ref.
[52]). Chu et al. [53] have shown that vaccination of mice with
egg lysozyme together with CpG oligonucleotide in IFA in-
duced a powerful Th1 immune response, comparable with that
achieved by injection of the antigen in CFA. Like CFA, bac-
terial DNA and synthetic CpG oligonucleotides were found to
cause extramedullary hematopoiesis in mice [54].
MECHANISMS OF ACTION
Most studies on the mechanism underlying the adjuvant effect
of CFA were conducted in the early years following its discov-
ery when little was known about the fate of peripherally in-
jected antigen. Freund had suggested three categories of action
mechanisms: 1) prolong[ation of] the presence of antigens at
the site of injection, 2) more effective “transport of the antigens
http://www.jleukbio.org
to the lymphatic system and to the lungs, where the adjuvant
promotes the accumulation of cells concerned with the immune
response,” and 3) other mechanisms that should remain un-
identified, because their clarification would require knowledge
about “how antibodies are formed and how sensitization devel-
ops” [1]. Dresser [55, 56] made an important observation when
he found that intravenous immunization of mice with an ag-
gregate-free, pure protein failed to elicit a primary response,
but the same intravenous injection given in association with
subcutaneously injected CFA did induce such a response. A
similar observation was made using a simple water-in-oil ad-
juvant and SRBC as the antigens [57], suggesting that not only
the mycobacterial component but also IFA exert adjuvant
action via a systemic pathway. Even today, however, the nature
of these mechanisms remains largely unresolved.
Enhancement of antigen uptake by APCs
It is generally assumed, although not documented by any study
of recent date, that administering protein antigens as a water-
in-oil emulsion prolongs their lifetime at the site of injection.
Estimations of local half-lifetime of such antigens have varied
considerably, with a highest value of approximately 3 months
[58]. This led to the original suggestion that a slow and even
release of antigen over a long period of time is perhaps the most
important mechanism of action of oil-in-water adjuvants. How-
ever, at the time it was made, this suggestion could not take
into account the fact that local DCs are main players in
determining the fate of the antigen. In line with their normal
function, immature DCs at the site of injection are supposed to
engulf particles of adjuvant-embedded antigen and transport
them to locoregional lymph nodes and undergo maturation into
fully active APCs that will present antigen fragments to T cells.
Tsuji et al. recently showed that maturation of human DCs was
enhanced by purified extract of BCG consisting of peptidogly-
can, arabinogalactan, and mycolic acids, supporting the idea
that an important in vivo function of CFA is precisely to
promote DC maturation [59]. Over time, however, as CFA and
antigen persist in the injected tissue, the number and function
of DCs may change. They can even assume down-regulatory
functions [60].
Overall enhancement of phagocytosis by CFA, but also by
IFA, was demonstrated in experiments measuring carbon
clearance from the peripheral blood [61]. Conceivably, phago-
cytic/pinocytic activity of DC is also enhanced in the presence
of IFA and CFA. The effect of CFA on further antigen traf-
ficking was analyzed in a series of studies done in the late
1960s, just following the discovery by Mitchell and Abbot in
1965 [62] that antigen injected without any adjuvant becomes
rapidly associated with the outer surface of the follicular DCs
in the lymph nodes. In rats, mice, and guinea pigs given
soluble antigens in CFA, enhanced production of antibody was
associated, not with such predominant follicular localization of
the antigen but with some sort of entrapment of the antigen in
the subcapsular sinuses of the draining lymph nodes [63– 65].
Similarly, in rats given IFA, the oil component was found to
accumulate selectively in the lymph nodes, first in the subcap-
sular sinuses and later in the cortex and paracortex [66]. In
mice given MF59, a metabolizable oil-in-water vaccine adju-
Billiau and Matthys Freund’s adjuvants in autoimmune diseases
vant, oil drops and antigen were found to follow the same
pathway. At 3 h after intramuscular injection, most of the
MF59 was still outside cells at the injection site, but some was
already detectable intracellularly in the subcapsular sinuses of
draining lymph nodes; at 48 h, adjuvant remaining in the
muscle was mostly inside cells bearing a DC cell marker.
Meanwhile, in the lymph nodes, the adjuvant had penetrated
the paracortex and cortex [67]. This is consistent with a sce-
nario in which adjuvant potentiates the pathway, whereby DCs
present antigen to T cells and generate an extra quotum of
T-cell help. However, the validity of this proposed scenario
needs further experimental support.
Emission of danger signals resulting
in Th1 skewing
Whereas IFA and CFA act as adjuvants for the production of
antibodies, CFA is essential for development of cell-mediated
responses such as DTH and certain experimental autoimmune
diseases. The mycobacteria in CFA are currently assumed to
be recognized by PAMP (pathogen-associated molecular pat-
tern) receptors on various immunocompetent cells and thus to
provide the stimulus for these cells to release mediators and
express membrane receptors (collectively designated as “dan-
ger” signals) that will lead to Th1-type skewing of ongoing
immune responses. In fact, immunization with IFA can, under
certain circumstances, lead to reduced cell-mediated re-
sponses. This was originally interpreted as tolerance but is now
seen as a Th2-directed skewing of the immune response. The
model holds that IFA, by failing to stimulate APCs for danger
signaling, favors development of a strong Th2-type response
[68, 69].
Cytokine induction
Little information is available on cytokine induction by adju-
vant only. In DA rats receiving IFA to induce OIA, mRNA for
TNF-␣ was found to be induced rapidly, with limited expres-
sion of IFN-␥ mRNA and no IL-2 mRNA. Remarkably, when
ovalbumin was added to the IFA, IL-4 mRNA rather than
TNF-␣ mRNA was detected [38], suggesting a deviation toward
Th2 responsiveness. Another study compared expression of
cytokine genes in lymph nodes and spleens of mice immunized
with collagen-II in CFA with expression in mice that received
the adjuvant alone [70]. In the two groups of mice, an identical
pattern of gene transcription, i.e., increased expression of IL-2
and IFN-␥ but undetectable levels of IL-4 and IL-5, was found.
Lymph node cells of mice immunized with collagen-II and CFA
responded to fragments of collagen-II, but their response to the
purified protein derivative (PPD) was much more pronounced.
For additional information on cytokine induction by killed
mycobacteria, we must rely on extrapolation from data obtained
with live organisms. The importance of two cytokines, IFN-␥
and TNF-␣, has been particularly well-documented. For in-
stance, Mycobacterium avium can grow exponentially in non-
activated murine macrophages, but this growth is restricted if
the macrophages are stimulated with IFN-␥ and TNF-␣ [71]. In
vivo, depletion of NK [72] or CD4 cells [71, 73] was shown to
result in enhanced proliferation of the organisms. At the same
853
time, expression of IFN-␥ and TNF-␣ mRNAs in the spleen
was reduced. In mice subjected to CD4-cell depletion (result-
ing in increased proliferation of mycobacteria) or to BCG
vaccination (resulting in reduced proliferation), splenic expres-
sion of the mRNAs of TNF-␣ and IFN-␥ was found to correlate
with ability to control proliferation. Furthermore, ablation ex-
periments using anti-TNF-␣ and anti-IFN-␣ monoclonal anti-
bodies indicated that both cytokines are important for early
protection and that IFN-␥ is involved in later T-cell-dependent
resistance [71]. Direct evidence that TNF-␣ and/or IFN-␥ are
produced locally after injection of CFA is not available, but the
presumption that this is the case is supported by a study on
dermal lesions caused by BCG in rabbits [74]. Within 1–5
days, mRNAs for both cytokines appeared at the injection site
(together with mRNA for IL-1␤, MCP-1, and IL-8). The authors
interpreted this early transient response as the result of a
nonspecific adjuvant-type effect of the mycobacteria present in
the BCG inoculum.
Another cytokine of great importance in the host response to
mycobacteria is IL-12, mainly produced by activated MPCs.
Optimal IL-12 production capacity in response to mycobacteria
requires pre-exposure by IFN-␥ [75]. In reverse, IFN-␥ pro-
duction is itself enhanced by IL-12, because IL-12 directly
activates NK cells to produce IFN-␥ and also directs prolifer-
ating helper T cells to assume an IFN-␥-secretory (Th1) profile.
In vivo ablation of IL-12 by treatments with neutralizing anti-
bodies [76, 77] or by the gene knock-out (KO) approach [78,
79] drastically reduces innate and acquired resistance as well
as immune reactivity against mycobacteria. It should be noted
that the innate TNF-␣ response, in contrast to the IFN-␥
response, seems to be independent of IL-12: Lung macro-
phages of BCG-infected IL-12 KO mice did release TNF-␣ but
not IFN-␥ [80].
IL-6 is another cytokine likely to be induced by CFA that
may be relevant for induction of autoimmune diseases. Direct
in vivo demonstration of IL-6 production following injection of
CFA is not available. Again, we have to rely on information
obtained with viable mycobacteria or some of their compone-
nents. Mycobacteria and their components induce IL-6 in
spleen-cell cultures of uninfected and, more so, of BCG-in-
fected mice [81]. The same goes for M. avium intracellulare
[82]. Murine bone marrow-derived MPCs infected with BCG
were found to secrete a factor, identifiable as IL-6, which
inhibits uninfected MPCs to function as APCs for antigen-
specific activation of T lymphocytes [83]; in this study, heat-
killed mycobacteria were found to be unable to induce this
factor. However, in other studies in human MPCs, muramyl
dipeptide and lipoarabinomannan were found to stimulate IL-6
production [84, 85].
What is a likely schedule of cytokine induction following
exposure to CFA, and how can these cytokines explain the role
of CFA as a facilitator of autoimmune disease? Early cytokine
induction is likely to be triggered mainly by the adjuvant and
less by the injected autoantigen. However, the cytokines con-
ceivably fulfill an up-regulatory role for antigen-specific T
cells, which will ultimately result in polyclonal activation of
these cells and possibly of bystanders as well. A proposed
scheme for this early up-regulatory network is represented in
854 Journal of Leukocyte Biology Volume 70, December 2001
Figure 1. As evident from the reviewed evidence, primary
target cells for the adjuvant components are MPCs and DCs,
which can produce TNF-␣, IL-12, and IL-6. Early IFN-␥ may
come from NK cells, which may become involved as soon as
IL-12 appears on the scene but may also be triggered more
directly through a pathway involving their activating receptor,
NKGD2, which recognizes MHC-I-like antigens induced on
several cells by stress signals [86]. IL-12 and IFN-␥ form a
positive feedback loop that potentiates deviation toward Th1-
type responsiveness of activated CD4⫹ T cells. Production of
TNF-␣ can be presumed to play a role as inducer of other
cytokines (such as IL-6) and chemokines. IL-6 may play a role
as stimulator of autoantibody production and activator of T
lymphocytes. In the later phases, as disease becomes overt,
long-lived CFA components may continue to stimulate cyto-
kine production, but activated lymphocytes conceivably now
play an increasingly important part. One effect of prolonged
cytokine production, in particular of IL-12 and IL-6, is to
generate a myelopoietic response, which at least in some
models, constitutes a disease-promoting factor [87]. IFN-␥
produced in this phase can still act as an up-regulator of
MPC-like effector cells and thus potentially play a disease-
promoting role. However, via another pathway, IFN-␥ can act
against disease by counteracting the myelopoietic effect of
CFA.
Chemokine induction
Mycobacteria may induce chemokine production directly by
the phagocytes in which they reside or via induction of cyto-
kines that induce other cells to produce chemokines. In a
human alveolar epithelial cell line (A549), infection with var-
ious strains of M. tuberculosis induced mRNAs and proteins of
Fig. 1. Cytokines observed to be induced in the early phases following
exposure to CFA (or mycobacteria) are TNF-␣, IL-12, IL-6, IFN-␥, and several
chemokines. Mycobacterial components are known to target MPCs and DCs
(involving toll-like receptors) and to induce production of monokines, in
particular IL-12 and TNF-␣. IL-12 induces NK cells to produce IFN-␥, which
potentiates production of IL-12, forming a positive feedback loop (curved
arrows). More direct stimulation of NK cells might take place via their
activating receptor NKGD2, which recognizes stress-induced membrane li-
gands. TNF-␣ can be presumed to play a role as inducer of other cytokines
(such as IL-6) and of chemokines. IL-12 is the driving force for directing T-cell
differentiation to assume a Th1 profile.
http://www.jleukbio.org
MCP-1 and IL-8 but not MIP-1␣ or RANTES (regulated on The significance of granuloma formation for the adjuvant and
activation, normal T expressed and secreted) [88]. Induction autoimmunity-promoting effects of CFA is unclear. It can be
seemed not to depend on prior induction of IL-6, TNF-␣, or presumed that the MPCs, which constitute a major part of the
IL-1␤, however heat-killed mycobacteria failed to induce. Con- cell population in granulomas, serve as an extra source of
versely, in human PBMCs, live mycobacteria (BCG) as well as cytokines, chemokines, and other inflammatory mediators and
lipoarabinomannan induced appearance of mRNA for MCP-3 may thus influence antigen presentation as well as lymphocyte
[48]. In mice treated with PPD-coated beads (vide supra) development and differentiation during the induction phase.
increased mRNA levels for MCP-1, MCP-3, and RANTES but
not for eotaxin were found in granulomatous lung tissue [89]. In Expansion and subsequent contraction of
cutaneous lesions in rabbits infected with BCG, mononuclear activated CD4⫹ T cells
cells were shown to contain mRNAs for MCP-1 or IL-8, the first
In mice infected with BCG or given a CFA-assisted immuni-
ones being found in areas with mononuclear-cell infiltration
zation schedule against myelin oligodendrocyte glycoprotein
and the second ones, in areas with neutrophil infiltration [74].
(MOG; for induction of EAE), a splenic population of activated
Other evidence for IL-8, the protoptype C-X-C chemokine, to
(CD44hi) CD4⫹ T cells was found to first expand and then
be involved was obtained in a study on alveolar macrophages
retract, the peak of the population size occurring 3.5 weeks
from patients with pulmonary tuberculosis in comparison with
after BCG or 2 weeks after CFA [96, 97]. In both instances, the
controls [90]. IL-8 protein release was elevated in lavage fluid
expansion was significantly increased and retraction signifi-
and in supernatants of cultured macrophages of patients; IL-8
cantly delayed in IFN-␥ KO mice. Moreover, in both cases
mRNA levels were also elevated. In vitro stimulation of mac-
retraction correlated with apoptosis. This and accompanying in
rophages with mycobacterial cell components induced release
vitro evidence indicated that in BCG-infected and in CFA-
of IL-8, which could be inhibited by antibodies against TNF-␣
treated mice, IFN-␥ is required for down-regulating expansion
and/or IL-1, suggesting that induction of IL-8 was indirect. On
of the activated T cells. In the BCG-infected mice, the T cells
the basis of this limited amount of available information, it is
were found to react against mycobacterial antigen (PPD); in
reasonable to assume that CFA induces production of C-C and
mice immunized against MOG in CFA, the cells reacted with
C-X-C chemokines at the sites where MPCs have engulfed the
MOG. Although reactivity against mycobacterial antigen was
heat-killed mycobacteria. However, a more detailed picture
not demonstrated for the latter ones, the analogy between the
and evaluation of the role of these chemokines in the effects of
two systems suggests that a large proportion (if not the larger
CFA must await further study.
one) of the cells may have had reactivity toward PPD and other
Granuloma formation mycobacterial antigens rather than for MOG. A consideration
not formulated by the authors is that in terms of mechanism by
In granulomatous lung tissue generated by preimmunizing the
which CFA promotes autoimmune disease, these mycobacterial
animals with PPD in CFA and then challenging them with
antigen-reactive, activated CD4⫹ cells may be as important in
PPD-coated beads, the mRNAs for IFN-␥ and TNF-␣ were
EAE pathogenesis as the MOG-reactive cells. Indeed, evi-
found to be detectable [89]. From studies with live Mycobac-
dence supporting a model for induction of EAE holds that any
terium bovis BCG, some information is available on the role of
activated T cell, irrespective of its antigen-specificity, tends to
cytokines in the pathogenesis of these granulomas. IFN-␥R KO
cross the blood brain barrier and enter the neuropil [98, 99].
mice, after inoculation of BCG, showed reduced formation of
We know that such cells rapidly undergo apoptosis, because
such lesions in the liver [91]. These mice were also less
they do not encounter the relevant antigens [100]. Neverthe-
efficient in controlling proliferation and spread of the bacteria
less, by their sheer number, they may contribute significantly
and died within weeks after infection. IFN-␥ ligand KO mice
to the initiation of inflammation in the central nervous system.
infected with virulent M. tuberculosis did develop granulomas
It was also shown that addition of IFN-␥ to cultured spleno-
but were nevertheless less able than wild-type mice in restrict-
cytes of BCG-infected or CFA-treated IFN-␥ KO mice resulted
ing growth of the mycobacteria [92]. From another study using
in apoptosis of CD4⫹ T cells and that at least in the first case,
IFN-␥ KO mice, it appeared that granuloma formation simi-
this apoptosis could be inhibited by depletion of adherent or
larly did not depend critically on the availability of IFN-␥ [93].
Mac-1⫹ cells. This led the authors to suggest a model in which
As a contrast, TNF-␣ seems to be indispensable for granuloma
T cells produce IFN-␥, which activates MPCs, which, in turn,
formation as is evident from observations using a neutralizing
cause apoptosis of the T cells [96, 97]. In this model, IFN-␥ is
antibody to TNF in mice infected with a BCG strain [94]; in
a link in a disease-controlling feedback loop.
mice treated with the antibody, the “organogenesis” of the liver
granulomas as well as their bactericidal function was sup- Haemopoietic dysfunction
pressed. Moreover, administration of the antibody caused ac-
celerated regression of established granulomas. Studies relying The well-known, protracted splenomegaly that develops in
on the use of TNF receptor I KO mice or of treatment with CFA-treated animals results from overall leukoproliferation
soluble TNF receptor I have led to the same conclusion [95]. together with accumulation of dispersed granulomas. The sig-
Extrapolating from these observations with live bacteria, one nificance of haemopoietic dysfunction as a factor underlying
may presume that granuloma formation after treatment with the diverse adjuvant and other functional biological effects of
CFA depends mainly on TNF-␣ production and only to a minor CFA has until recently escaped the attention of investigators.
extent on IFN-␥. In fact, the number of studies that specifically address the

Billiau and Matthys Freund’s adjuvants in autoimmune diseases 855

effect of CFA on haematopoiesis is limited, and we have to
consider whatever information is available together with that
from studies done with live bacteria.
In the 1960s and 1970s, “vaccination” with BCG was stud-
ied experimentally and clinically for its ability to augment host
defense against leukemia and several other tumors. In these
systems, one aspect of the mode of action of BCG was found to
be its ability to enhance repopulation of bone marrow and
leukopoiesis after myeloablative chemotherapy [101]. Pretreat-
ment of mice with BCG or CFA was shown to condition the
animals for recovery of leukopoietic functions following a sub-
sequent myelosuppressive cyclophosphamide bolus injection.
Treatment with BCG or CFA alone was also shown to cause
increases in colony-forming units in bone marrow and in levels
of CSFs in serum [101]. Similarly, mycobacterium-related im-
munoadjuvants of defined chemical nature were also found to
exert haemopoietic effects. For instance, MDP given to mice
induced a rise in the level of monocyte-macrophage colony-
stimulating activity in serum, expansion of GM progenitors in
the spleen, and proliferation of multipotential stem cells in
bone marrow [41]. Other examples are the stimulatory effect of
a glycopeptidolipid fraction of Mycobacterium chelonae [102]
and of TDM [103] on repopulation of the haemopoietic system
in irradiated mice. The mechanisms underlying these haema-
topoietic effects have not been studied in much detail. Little is
known, for example, about the spectrum or the cellular origin
of the haematopoietic cytokines involved.
However, much can be learned from recent studies showing
that the antimycobacterial response is exaggerated in mice with
a defective IFN-␥ system. BCG infection was shown to cause
more profound hematopoietic alterations in IFN-␥R KO than in
wild-type mice [104]; this was accompanied by higher levels of
hematopoietic cytokines IL-6, IL-3, and G-CSF. The combina-
tion of overall increased extramedullary haematopoiesis and
granuloma formation resulted in complete disruption of the
normal splenic histological architecture. Also, in murine ar-
thritis models, which rely on the use of CFA for autoimmuni-
zation with collagen, enhanced bone marrow myleopoiesis and
extramedullary haematopoiesis were noted to occur [9, 105].
Like in BCG infection, this remodeling of the haematopoietic
system was much more pronounced in IFN-␥R KO mice than
in the wild-type counterparts [9], indicating that whatever
myelopoietic factors are involved, the role of IFN-␥ consists of
restraining their actions. Enhanced myelopoiesis in IFN-␥R
KO mice given CFA could be suppressed by treatment with
neutralizing anti-IL-12 or anti-IL-6 antibodies [9], suggesting
that both these cytokines are involved as myelopoietic factors.
In mice treated with CFA to induce CIA, the cell population
most affected by haemopoietic remodeling was that of imma-
ture Mac-1⫹ myeloid cells, i.e., precursors to MPCs and neu-
trophils. In the spleen, the total cellularity and the proportion
of Mac-1 positives were augmented, and peak levels coincided
with appearance of the first arthritis symptoms [9]. Increased
myelopoiesis in the IFN-␥R KO mice was associated with
higher disease scores, more intense DTH to collagen-II, and a
more distinct Th1 profile of cytokines induced by an in vivo
challenge with anti-CD3 antibody. Treatment with anti-IL-12
or anti-IL-6 antibody resulted in reduced expansion of the
856 Journal of Leukocyte Biology Volume 70, December 2001
Mac-1⫹ population and in lower disease scores. Accordingly, it
was suggested that these Mac-1⫹ cells constitute the pool of
effectors for the tissue damage occurring in CIA and possibly
in other CFA-based experimental models of autoimmune dis-
ease [87, 106].
CONCLUSIONS
For more than 50 years, immunologists have used CFA to
induce autoimmune diseases in experimental animals. In re-
viewing the literature, one is struck that, especially in the last
decades, they have done so with almost complete disregard of
the mechanisms by which CFA intervenes in these experimen-
tal situations. In interpretations of experimental data, the pos-
sible role of CFA has rarely been a point of discussion. In our
review, we have made it clear that this should indeed have
been an issue. Our review also indicates that too few studies
have been done specifically addressing biological effects of
CFA as such. As a consequence, to conceptualize what the role
of CFA in these models could be, we must rely on information
derived from studies conducted for different and disparate
purposes, not the least from ones on infection with live myco-
bacteria. Yet, by collating evidence from these studies, we can
assemble a coherent picture that takes into account the rele-
vant contemporary concepts on the function of the immune
system.
In the early years of work with Freund’s adjuvant, three
categories of action mechanisms were proposed. Two of these
remain fully valid: the longer lifetime of autoantigens injected
in IFA or CFA and their altered trafficking to critical sites in
the immune system. Of note, these two mechanisms apply to
IFA as well as to CFA, because they both result from embed-
ding the antigen in an oily excipient. Uptake of antigens by
APCs and subsequent trafficking of these cells are currently
hot subjects of investigation in immunology. Relevant networks
of cells and molecules, in particular the chemokines and their
receptors, are in the process of being untangled. How the
administration of IFA and CFA affects these networks at a
molecular and cellular basis is, unfortunately, not yet a major
point of concern.
The third category of mechanisms, which the early workers
had to leave largely “unidentified,” can now at least be par-
tially specified and accommodated into a plausible scenario
that involves mainly mechanisms of innate immunity. IFA and
CFA are good at activating these mechanisms, and in doing so,
direct and orchestrate development and function of antigen-
specific T and B lymphocytes. The framework of the proposed
scenario is shown in Figure 2. The oil component and, more
so, the mycobacteria, activate the MPC system, including the
various categories of DCs. This results in overall enhanced
phagocytosis of particulate material [61] and secretion of
monokines [38], a correlate of this being the transient, in-
creased aspecific enhancement of resistance to infection [37].
This, in turn, results in stronger-than-normal polyclonal acti-
vation and proliferation of T lymphocytes, which, regardless of
their being autoantigen-specific but precisely as a result of
their status of being activated, tend to infiltrate into tissues
http://www.jleukbio.org

Fig. 2. Synopsis of action mechanisms of Freund’s adjuvants in facilitating
experimental autoimmune disease (see Conclusions). Being suspended in oil,
injected antigens and mycobacteria have a longer overall in vivo lifetime and
are differently trafficked throughout the lymphoid system. Three consecutive
levels (top to bottom) of responding systems are shown: MPCs (including DCs),
T lymphocytes (both reacting over a period of days to weeks), and the
haemopoietic system (reacting after weeks). Effects of mineral oil (left, green)
and mycobacteria (CFA; right, red) are largely overlapping; IFA favors Th2-
type responsiveness of T lymphocytes, whereas CFA favors Th1 responsive-
ness. The left text column lists observations testifying to the effects that are
represented in the figure (see also Conclusions).
despite physical impediments such as the blood-brain barrier
[39, 98, 99]. The activated T lymphocytes also produce sets of
lymphokines, representing a type 1 or type 2 helper activity
depending on whether the immunization was done with protein
antigen only (i.e., antigen in IFA) or with antigen in conjunc-
tion with mycobacteria (i.e., antigen in CFA) [68, 69]. In both
instances, an increased antibody response is generated, but
DTH develops preferentially in the second instance.
Until this stage, one could say that for inducing autoimmu-
nity and autoimmune disease, Freund adjuvants use the same
mechanisms as those by which they enhance specific immune
responses to foreign antigens. However, in the case of CFA, the
heat-killed mycobacterial cells embedded in oily excipient
persist for weeks or even months at the injection site and in
phagocyte-rich organs such as lung and liver. As time elapses,
they thus constitute an unabated stimulus for the production of
monokines and Th1 lymphokines. These late-produced cyto-
kines may play a role in bringing about the arrest of T-cell
expansion and activation but also in the gradual build-up of
Billiau and Matthys Freund’s adjuvants in autoimmune diseases
myelopoiesis, most explicitly revealing itself in splenomegaly
and disruption of spleen histology. The Mac-1⫹ cells generated
by this haemopoietic activity can fulfill different roles in fur-
ther evolution of the disease. On the one hand, they may
constitute a source of additional effector cells that after matu-
ration and activation, add to the tissue damage. Conversely,
there is evidence for the emerging Mac-1⫹ population to act as
suppressor of T-cell activation [34]. Which of these opposing
roles predominate may depend on the time of their emergence
relative to the other events in disease pathogenesis. In CIA,
CFA-induced myelopoietic activity and emergence of Mac-1⫹
correlate with disease activity [9, 87]. In other models, how-
ever, such as diabetes in NOD mice, the opposite is the case.
Polyclonal activation of lymphocytes is probably the crucial
event in models where administration of IFA or CFA without
intentionally added autoantigen causes autoimmune disease,
notably OIA and AIA. To explain induction of AIA by myco-
bacterium-containing CFA, cross-reactivity of antimycobacte-
rial antibodies or T-cell receptors with epitopes of host proteins
have been invoked. Yet the identity of these epitopes so far
remains unknown, and other mechanisms may need to be
considered. In the case of OIA, no proteinaceous antigen is
used; nevertheless, the disease is T-cell-mediated, suggesting
that arthritogenic T-cell clones are constitutively present
whose pathogenic activity is only marginally controlled by
mechanisms of peripheral tolerance. Induction of disease by oil
adjuvant could then be a result of abrogation of such tolerance.
Understanding how Freund’s adjuvants facilitate induction
of experimental autoimmune disease allows us to define crucial
elements in the pathogenesis of these models and, by extrap-
olation, in the naturally occurring human diseases. Whereas,
intuitively, one would tend to explain the role of adjuvants by
their facilitating effect on the generation of autoantigen-recog-
nizing lymphocyte clones, the scenario evoked here rather
stresses the importance of increased aspecific activation of
MPCs, DCs, and T lymphocytes and the excess generation of
aspecific effector and regulator cells. The implication is that
these mechanisms may also deserve more attention in deci-
phering the mechanisms of emergence, remission, and recru-
descence of natural autoimmune diseases in man. Further
research on the cellular and molecular mechanisms underlying
adjuvant action in experimental models is therefore warranted.
ACKNOWLEDGMENTS
Work in the authors’ laboratory is supported by grants from the
Fund for Medical Scientific Research of Flanders (FWO), from
the Regional Government of Flanders (GOA program), and
from the Belgian Federal Government (IUAP program). P. M. is
a postdoctoral research fellow of the FWO.
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