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Rega Institute, University of Leuven, Belgium
Correspondence: A. Billiau, Rega Institute, University of Leuven, Laboratory of Immunobiology, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail: Alfons.Billiau{at}Rega.Kuleuven.ac.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONBRIEF HISTORYACTIONS OF CFATHE ACTIVE COMPONENTSMECHANISMS OF ACTIONCONCLUSIONSREFERENCES |
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Key Words: Freund • adjuvant • dendritic cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONBRIEF HISTORYACTIONS OF CFATHE ACTIVE COMPONENTSMECHANISMS OF ACTIONCONCLUSIONSREFERENCES |
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BRIEF HISTORY |
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TOPABSTRACTINTRODUCTIONBRIEF HISTORYACTIONS OF CFATHE ACTIVE COMPONENTSMECHANISMS OF ACTIONCONCLUSIONSREFERENCES |
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A second line of investigation began with the observation that guinea pigs infected with M. tuberculosis respond differently 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 hypersensitivity 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 "tuberculin sensitiveness" (i.e., time course, characteristic superficial 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 infected 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 mycobacteria 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" complement-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, experimental autoimmune orchitis, experimental autoimmune thyroiditis, CIA, and myasthenia gravis.
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ACTIONS OF CFA |
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TOPABSTRACTINTRODUCTIONBRIEF HISTORYACTIONS OF CFATHE ACTIVE COMPONENTSMECHANISMS OF ACTIONCONCLUSIONSREFERENCES |
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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 susceptible rats or mice [14 ].
Adjuvant-induced arthritis (AIA), inducible by CFA (without 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 pathogenesis 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 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 restrictions [20
21
22
]. The
reason is that within days after the subcutaneous administration of
CFA, a strong and long-lasting inflammatory 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, hyperplasia
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. Occasionally, 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 population. Although
there appears to have been no explicit follow-up of this observation,
infection of mice with Mycobacterium lepraemurium has been
shown to result in similar generation 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 precursors in the infected mouse, 2)
maturation upon overnight in vitro culture, and 3) activation following
exposure to the Con A-stimulated indicator splenocytes. Stage 1, in
vivo, was found to depend on activity of sensitized lymphocytes; stage
2 required the presence of a nonadherent, non-T, non-B, non-natural
killer (NK) cell population; and stage 3 was distinguishable 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 responses of maternal splenocytes
toward paternal allo-antigens, suppressive effect of lymphocytes
derived from spleen, lymph nodes, or placenta for maternal lymphocyte
responses to paternal 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 development 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 recipients 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
].
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THE ACTIVE COMPONENTS |
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TOPABSTRACTINTRODUCTIONBRIEF HISTORYACTIONS OF CFATHE ACTIVE COMPONENTSMECHANISMS OF ACTIONCONCLUSIONSREFERENCES |
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(TNF-) in regional lymph
nodes [38
], and causes opening of the blood-brain
barrier [39
]. The adjuvant effect of IFA on the immune
response to SRBC can be blocked by administration of antioxidants,
suggesting that IFA induces production of oxygen radicals
[40
]. IFA can also trigger autoimmune-like disease, in
particular, arthritis, in genetically predisposed animals (vide supra). An intensely investigated question is the chemical nature of the mycobacterial substances that account for the immunoadjuvant 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 incorporated in a water-oil emulsion. One aspect of the adjuvant effect of these substances is their ability to stimulate haematopoiesis. 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-macrophage progenitors in the spleen, and proliferation of multipotential stem cells in bone marrow [41 ].
Glycolipids typical for Mycobacteria are trehalose dimycolate (TDM) and lipoarabinomannan. Mycolic acids are long-chain branched ß-hydroxy carboxylic acids that occur in Mycobacteria, 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 lipopolysaccharides (LPS).
Lipoarabinomannans (LAM) consist of a cell membrane-bound lipid core,
phosphatidylinositol, carrying a phosphodiester-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 polysaccharide 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. Nevertheless, 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 trafficked back to the cell surface in association with
CD1 and presented as a major histocompatibility complex
(MHC)-independent 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 chemoattractant 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 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 increased 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 possessing 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 initiation of autoimmunity. However, clinical and experimental evidence have indicated an opposite effect of HSPs [18 ]. Experimental immunization with HSPs leads mostly to a resistance to subsequent induction of autoimmune disease. Moreover, 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 mycobacteria, 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 induced a powerful Th1 immune response, comparable with that achieved by injection of the antigen in CFA. Like CFA, bacterial DNA and synthetic CpG oligonucleotides were found to cause extramedullary hematopoiesis in mice [54 ].
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MECHANISMS OF ACTION |
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TOPABSTRACTINTRODUCTIONBRIEF HISTORYACTIONS OF CFATHE ACTIVE COMPONENTSMECHANISMS OF ACTIONCONCLUSIONSREFERENCES |
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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. However, 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 peptidoglycan, 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, phagocytic/pinocytic activity of DC is also enhanced in the presence of IFA and CFA. The effect of CFA on further antigen trafficking 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 64 65 ]. Similarly, in rats given IFA, the oil component was found to accumulate selectively in the lymph nodes, first in the subcapsular sinuses and later in the cortex and paracortex [66 ]. In mice given MF59, a metabolizable oil-in-water vaccine adjuvant, 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 scenario 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 pattern) receptors on various
immunocompetent cells and thus to provide the stimulus for these cells
to release mediators and express membrane receptors (collectively
designated as "danger" 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 responses. 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
adjuvant only. In DA rats receiving IFA to induce OIA, mRNA for TNF-
was found to be induced rapidly, with limited expression 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 instance,
Mycobacterium avium can grow exponentially in nonactivated
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 time, expression of IFN- and TNF- mRNAs in
the spleen was reduced. In mice subjected to CD4-cell depletion
(resulting in increased proliferation of mycobacteria) or to BCG
vaccination (resulting in reduced proliferation), splenic expression of
the mRNAs of TNF- and IFN- was found to correlate with ability to
control proliferation. Furthermore, ablation experiments using
anti-TNF- and anti-IFN- monoclonal antibodies 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-1ß, 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- production is
itself enhanced by IL-12, because IL-12 directly activates NK cells to
produce IFN- and also directs proliferating helper T cells to assume
an IFN--secretory (Th1) profile. In vivo ablation of IL-12 by
treatments with neutralizing antibodies [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 macrophages 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 componenents. Mycobacteria and their components induce IL-6 in spleen-cell cultures of uninfected and, more so, of BCG-infected 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 conceivably 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 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 cytokine 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.
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or RANTES (regulated on activation, normal T expressed and
secreted) [88
]. Induction seemed not to depend on prior
induction of IL-6, TNF-, or IL-1ß, however heat-killed
mycobacteria failed to induce. Conversely, in human PBMCs, live
mycobacteria (BCG) as well as lipoarabinomannan induced appearance of
mRNA for MCP-3 [48
]. In mice treated with PPD-coated
beads (vide supra) increased mRNA levels for MCP-1, MCP-3, and RANTES
but not for eotaxin were found in granulomatous lung tissue
[89
]. In cutaneous lesions in rabbits infected with BCG,
mononuclear cells were shown to contain mRNAs for MCP-1 or IL-8, the
first ones being found in areas with mononuclear-cell infiltration and
the second ones, in areas with neutrophil infiltration
[74
]. Other evidence for IL-8, the protoptype C-X-C
chemokine, to be involved was obtained in a study on alveolar
macrophages from patients with pulmonary tuberculosis in comparison
with controls [90
]. IL-8 protein release was elevated in
lavage fluid and in supernatants of cultured macrophages of patients;
IL-8 mRNA levels were also elevated. In vitro stimulation of
macrophages with mycobacterial cell components induced release of IL-8,
which could be inhibited by antibodies against TNF- and/or IL-1,
suggesting that induction of IL-8 was indirect. On the basis of this
limited amount of available information, it is reasonable to assume
that CFA induces production of C-C and C-X-C chemokines at the sites
where MPCs have engulfed the heat-killed mycobacteria. However, a more
detailed picture and evaluation of the role of these chemokines in the
effects of CFA must await further study.
Granuloma formation
In granulomatous lung tissue generated by preimmunizing the
animals with PPD in CFA and then challenging them with PPD-coated
beads, the mRNAs for IFN- and TNF- were found to be detectable
[89
]. From studies with live Mycobacterium
bovis BCG, some information is available on the role of cytokines
in the pathogenesis of these granulomas. IFN-R KO mice, after
inoculation of BCG, showed reduced formation of such lesions in the
liver [91
]. These mice were also less efficient in
controlling proliferation and spread of the bacteria and died within
weeks after infection. IFN- ligand KO mice infected with virulent
M. tuberculosis did develop granulomas but were nevertheless
less able than wild-type mice in restricting growth of the mycobacteria
[92
]. From another study using IFN- KO mice, it
appeared that granuloma formation similarly did not depend critically
on the availability of IFN- [93
]. As a contrast,
TNF- seems to be indispensable for granuloma formation as is evident
from observations using a neutralizing antibody to TNF in mice infected
with a BCG strain [94
]; in mice treated with the
antibody, the "organogenesis" of the liver granulomas as well as
their bactericidal function was suppressed. Moreover, administration of
the antibody caused accelerated regression of established granulomas.
Studies relying on the use of TNF receptor I KO mice or of treatment
with soluble TNF receptor I have led to the same conclusion
[95
]. Extrapolating from these observations with live
bacteria, one may presume that granuloma formation after treatment with
CFA depends mainly on TNF- production and only to a minor extent on
IFN-.
The significance of granuloma formation for the adjuvant and autoimmunity-promoting effects of CFA is unclear. It can be presumed that the MPCs, which constitute a major part of the cell population in granulomas, serve as an extra source of cytokines, chemokines, and other inflammatory mediators and may thus influence antigen presentation as well as lymphocyte development and differentiation during the induction phase.
Expansion and subsequent contraction of activated CD4+
T cells
In mice infected with BCG or given a CFA-assisted immunization
schedule against myelin oligodendrocyte glycoprotein (MOG; for
induction of EAE), a splenic population of activated
(CD44hi) CD4+ T cells was found to first expand
and then retract, the peak of the population size occurring 3.5 weeks
after BCG or 2 weeks after CFA [96
, 97
]. In
both instances, the expansion was significantly increased and
retraction significantly delayed in IFN- KO mice. Moreover, in both
cases retraction correlated with apoptosis. This and accompanying in
vitro evidence indicated that in BCG-infected and in CFA-treated mice,
IFN- is required for down-regulating expansion of the activated T
cells. In the BCG-infected mice, the T cells were found to react
against mycobacterial antigen (PPD); in mice immunized against MOG in
CFA, the cells reacted with MOG. Although reactivity against
mycobacterial antigen was not demonstrated for the latter ones, the
analogy between the two systems suggests that a large proportion (if
not the larger one) of the cells may have had reactivity toward PPD and
other mycobacterial antigens rather than for MOG. A consideration not
formulated by the authors is that in terms of mechanism by which CFA
promotes autoimmune disease, these mycobacterial antigen-reactive,
activated CD4+ cells may be as important in EAE
pathogenesis as the MOG-reactive cells. Indeed, evidence supporting a
model for induction of EAE holds that any activated T cell,
irrespective of its antigen-specificity, tends to cross the blood
brain barrier and enter the neuropil [98
,
99
]. We know that such cells rapidly undergo apoptosis,
because they do not encounter the relevant antigens
[100
]. Nevertheless, by their sheer number, they may
contribute significantly to the initiation of inflammation in the
central nervous system.
It was also shown that addition of IFN- to cultured splenocytes of
BCG-infected or CFA-treated IFN- KO mice resulted in apoptosis of
CD4+ T cells and that at least in the first case, this
apoptosis could be inhibited by depletion of adherent or
Mac-1+ cells. This led the authors to suggest a model in
which T cells produce IFN-, which activates MPCs, which, in turn,
cause apoptosis of the T cells [96
, 97
]. In
this model, IFN- is a link in a disease-controlling feedback loop.
Haemopoietic dysfunction
The well-known, protracted splenomegaly that develops in
CFA-treated animals results from overall leukoproliferation together
with accumulation of dispersed granulomas. The significance of
haemopoietic dysfunction as a factor underlying the diverse adjuvant
and other functional biological effects of CFA has until recently
escaped the attention of investigators. In fact, the number of studies
that specifically address the 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 studied 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 ]. Pretreatment of mice with BCG or CFA was shown to condition the animals for recovery of leukopoietic functions following a subsequent 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 immunoadjuvants 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 haematopoietic 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 combination of
overall increased extramedullary haematopoiesis and granuloma formation
resulted in complete disruption of the normal splenic histological
architecture. Also, in murine arthritis models, which rely on the use
of CFA for autoimmunization 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 immature
Mac-1+ myeloid cells, i.e., precursors to MPCs and
neutrophils. 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 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 disease [87
,
106
].
|
CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONBRIEF HISTORYACTIONS OF CFATHE ACTIVE COMPONENTSMECHANISMS OF ACTIONCONCLUSIONSREFERENCES |
|---|
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 embedding 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 partially 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, increased aspecific enhancement of resistance to infection [37 ]. This, in turn, results in stronger-than-normal polyclonal activation 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 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 conjunction 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.
|
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 mycobacterium-containing CFA, cross-reactivity of antimycobacterial 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 extrapolation, 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-recognizing 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 deciphering the mechanisms of emergence, remission, and recrudescence of natural autoimmune diseases in man. Further research on the cellular and molecular mechanisms underlying adjuvant action in experimental models is therefore warranted.
|
ACKNOWLEDGEMENTS |
|---|
Received February 26, 2001; revised August 30, 2001; accepted September 13, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONBRIEF HISTORYACTIONS OF CFATHE ACTIVE COMPONENTSMECHANISMS OF ACTIONCONCLUSIONSREFERENCES |
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