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(Journal of Leukocyte Biology. 2001;70:849-860.)
© 2001 by Society for Leukocyte Biology

Modes of action of Freund’s adjuvants in experimental models of autoimmune diseases

Alfons Billiau and Patrick Matthys

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
 
Freund’s adjuvants are irreplaceable components of induction protocols of many experimental 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 prolonging the lifetime of injected autoantigen, by stimulating its effective delivery to the immune system and by providing a complex set of signals to the innate compartment of the immune system, resulting in altered leukocyte proliferation and differentiation. 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 CD4+ lymphocytes. The mycobacterial components within CFA signal T lymphocytes to assume a Th1 profile so that strong delayed-type hypersensitivity against autoantigens develops. In the absence of mycobacteria, T-lymphocyte differentiation tends to assume a Th2 profile with strong antibody production only. The mycobacterial 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-1+ 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 compartment 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.

Key Words: Freund • adjuvant • dendritic cells


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INTRODUCTION
 
For half a century, incomplete (IFA) and complete Freund’s adjuvant (CFA) have been the most commonly used immunoadjuvants 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 continued 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 autoimmune disease, such as experimental autoimmune encephalomyelitis (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.


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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 antigens 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. Injections 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 nonpathogenic 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 experimental animals caused lesions at the sites of injection somewhat resembling tubercles, but when the bacteria were incorporated into butter, they evoked a cellular reaction almost identical with a tubercle caused by pathogenic tubercle bacilli... ," 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 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
 
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-emulsified 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 considerably 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 pertussis vaccine for optimal reproducibility. Even in highly susceptible 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 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-{gamma} (IFN-{gamma}).

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
 
The combination of paraffin oil and surfactant, the two components of IFA, is in no way immunologically or pharmacologically 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 cytokines, predominantly tumor necrosis factor {alpha} (TNF-{alpha}) 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-{alpha}, 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
 
Most studies on the mechanism underlying the adjuvant effect of CFA were conducted in the early years following its discovery when little was known about the fate of peripherally injected 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 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 unidentified, because their clarification would require knowledge about "how antibodies are formed and how sensitization develops" [1 ]. Dresser [55 , 56 ] made an important observation when he found that intravenous immunization of mice with an aggregate-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 adjuvant 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. 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-{alpha} was found to be induced rapidly, with limited expression of IFN-{gamma} mRNA and no IL-2 mRNA. Remarkably, when ovalbumin was added to the IFA, IL-4 mRNA rather than TNF-{alpha} 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-{gamma} 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-{gamma} and TNF-{alpha}, 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-{gamma} and TNF-{alpha} [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-{gamma} and TNF-{alpha} 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-{alpha} and IFN-{gamma} was found to correlate with ability to control proliferation. Furthermore, ablation experiments using anti-TNF-{alpha} and anti-IFN-{alpha} monoclonal antibodies indicated that both cytokines are important for early protection and that IFN-{gamma} is involved in later T-cell-dependent resistance [71 ]. Direct evidence that TNF-{alpha} and/or IFN-{gamma} 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-{gamma} [75 ]. In reverse, IFN-{gamma} production is itself enhanced by IL-12, because IL-12 directly activates NK cells to produce IFN-{gamma} and also directs proliferating helper T cells to assume an IFN-{gamma}-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-{alpha} response, in contrast to the IFN-{gamma} response, seems to be independent of IL-12: Lung macrophages of BCG-infected IL-12 KO mice did release TNF-{alpha} but not IFN-{gamma} [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-{alpha}, IL-12, and IL-6. Early IFN-{gamma} 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-{gamma} form a positive feedback loop that potentiates deviation toward Th1-type responsiveness of activated CD4+ T cells. Production of TNF-{alpha} 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-{gamma} 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-{gamma} can act against disease by counteracting the myelopoietic effect of CFA.



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  		Figure 1. Cytokines observed to be induced in the early phases following exposure to CFA (or mycobacteria) are TNF-{alpha}, IL-12, IL-6, IFN-{gamma}, 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-{alpha}. IL-12 induces NK cells to produce IFN-{gamma}, 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 ligands. TNF-{alpha} 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.

Chemokine induction
Mycobacteria may induce chemokine production directly by the phagocytes in which they reside or via induction of cytokines that induce other cells to produce chemokines. In a human alveolar epithelial cell line (A549), infection with various strains of M. tuberculosis induced mRNAs and proteins of MCP-1 and IL-8 but not MIP-1{alpha} or RANTES (regulated on activation, normal T expressed and secreted) [88 ]. Induction seemed not to depend on prior induction of IL-6, TNF-{alpha}, 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-{alpha} 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-{gamma} and TNF-{alpha} 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-{gamma}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-{gamma} 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-{gamma} KO mice, it appeared that granuloma formation similarly did not depend critically on the availability of IFN-{gamma} [93 ]. As a contrast, TNF-{alpha} 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-{alpha} production and only to a minor extent on IFN-{gamma}.

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-{gamma} 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-{gamma} 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-{gamma} to cultured splenocytes of BCG-infected or CFA-treated IFN-{gamma} 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-{gamma}, which activates MPCs, which, in turn, cause apoptosis of the T cells [96 , 97 ]. In this model, IFN-{gamma} 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-{gamma} system. BCG infection was shown to cause more profound hematopoietic alterations in IFN-{gamma}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-{gamma}R KO mice than in the wild-type counterparts [9 ], indicating that whatever myelopoietic factors are involved, the role of IFN-{gamma} consists of restraining their actions. Enhanced myelopoiesis in IFN-{gamma}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-{gamma}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 ].


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CONCLUSIONS
 
For more than 50 years, immunologists have used CFA to induce autoimmune diseases in experimental animals. In reviewing 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 experimental situations. In interpretations of experimental data, the possible 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 mycobacteria. Yet, by collating evidence from these studies, we can assemble a coherent picture that takes into account the relevant 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 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.



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  		Figure 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 responsiveness. The left text column lists observations testifying to the effects that are represented in the figure (see also Conclusions).

Until this stage, one could say that for inducing autoimmunity 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 cytokines may play a role in bringing about the arrest of T-cell expansion and activation but also in the gradual build-up of 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 further evolution of the disease. On the one hand, they may constitute a source of additional effector cells that after maturation 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, however, 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 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.


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ACKNOWLEDGEMENTS
 
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.

Received February 26, 2001; revised August 30, 2001; accepted September 13, 2001.


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