Modes of action of Freund’s adjuvants in experimental models of autoimmune diseases (1)
incomplete (IFA) and complete Freund’s adjuvant (CFA) have been the most commonly used immuno adjuvants for many experimental models of autoimmune disease, such as experimental autoimmune encephalomyelitis (EAE), neuritis (EAN), uveitis (EAU), thyroiditis (EAT), and orchitis. IFA is essentially paraffin oil containing mannide mono-oleate as a surfactant. In addition, CFA contains heat-killed mycobacteria (Mycobacterium tuberculosis or other) and is more potent and more adequate for certain purposes.
ACTIONS OF CFA
Adjuvancity: hyperimmunization and facilitation of autoimmune disease induction
Experimental protocols for induction of autoimmune disease vary considerably depending on the target organ concerned and on the animal species or strain. For instance, in certain strains of rats, EAE can be induced without CFA, whereas in guinea pigs and in mice, CFA is mandatory, CFA is less crucial. Immunization of rats with collagen-II in IFA results in a form of protracted polyarthritis called “collagen-induced arthritis”. Adjuvant-induced arthritis (AIA), inducible by CFA (with- out added autoantigen) in rats, is a chronic disease. In Lewis rats, it develops in two phases: an acute periarticular inflammation followed by a phase of bone involvement.
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). This suppressor activity was associated with a nonadherent effector cell population. 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, or on the mitotic response of indicator lymphocytes to Con A.
THE ACTIVE COMPONENTS
The combination of paraffin oil and surfactant, the two components of IFA, is in no way immunologically or pharmacologically inert. IFA by itself exerts various effects on the immune system, locoregionally and systemically. Thus, IFA stimulates innate immunity, and induces expression of cytokines, predominantly tumor necrosis factor (TNF) in regional lymph nodes, and causes opening of the blood-brain barrier. The mycobacterial substances account for the immunoadjuvant effects of CFA. In CFA components, muramyl dipeptide (MDP) is a universally occurring building block of the peptidoglycan component of the bacterial cell wall. Glycolipids typical for Mycobacteria are trehalose dimycolate (TDM) and lipoarabinomannan. Lipoarabinomannans (LAM) consist of a cell membrane- bound lipid core, phosphatidylinositol, carrying a phosphodiester-bonded heteropolysaccharide chain containing arabinose and mannose residues. 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, and monocyte che- moattractant protein (MCP)-3. The mycobacterial heat shock proteins (HSPs) are other mycobacterial components that may play a role in the biological effects of CFA. Human hsp60, in particular, binds to and activates the toll-like receptor, Tlr4, resulting in an LPS-like effect on mononuclear phagocytes. However, clinical and experimental evidence have indicated an opposite effect of HSPs. Experimental immunization with HSPs leads mostly to a resis- tance to subsequent induction of autoimmune disease. CpG oligodeoxynucleotides, present in the heat-killed mycobacteria, may participate in bringing about the biological effects of CFA. 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.
MECHANISMS OF ACTION
Three categories of action mechanisms were suggested: 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”.
Enhancement of antigen uptake by APCs
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. 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.
Emission of danger signals resulting in Th1 skewing
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. The model holds that IFA, by failing to stimulate APCs for danger signaling, favors development of a strong Th2‐type response.
Cytokine induction
Direct evidence that TNF‐α and/or IFN‐γ are produced locally after injection of CFA is not available. 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. In human MPCs, muramyl dipeptide and lipoarabinomannan were found to stimulate IL‐6 production. Primary target cells for the adjuvant components are MPCs and DCs, which can produce TNF‐α, IL‐12, and IL‐6. IL‐12 and IFN‐γ form a positive feedback loop that potentiates deviation toward Th1‐type responsiveness of activated CD4+ T cells. Production of TNF‐α 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.
Chemokine induction
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‐1α or RANTES (regulated on activation, normal T expressed and secreted). Induction seemed not to depend on prior induction of IL‐6, TNF‐α, or IL‐1β, however heat‐killed mycobacteria failed to induce.
Granuloma formation
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.
Haemopoietic dysfunction
Develops in CFA‐treated animals results from overall leukoproliferation together with accumulation of dispersed granulomas. 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.
CONCLUSIONS
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 and secretion of monokines. Results in stronger‐than‐normal polyclonal activation and proliferation of T lymphocytes.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). In both instances, an increased antibody response is generated, but DTH develops preferentially in the second instance. In CIA, CFA‐induced myelopoietic activity and emergence of Mac‐1+ correlate with disease activity. In other models, however, such as diabetes in NOD mice, the opposite is the case.
