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. Introduction . Type I and Type II T-independent Antigens . Polyclonal B-lymphocyte Activation . B-cell Subpopulations . Immunological Tolerance to Thymus-independent Antigens . Genetics of Lipopolysaccharide Responsiveness: The Toll Family Genes . Immunoglobulin Cross-linking by Repeating Determinants . Lipopolysaccharide Receptors in Mammals . Pattern-recognizing Receptors and Pathogenassociated Molecular Patterns . Pattern-recognizing Receptors for Thymusindependent Antigens . PRRs, PAMPs and T Lymphocytes
Introduction
In most immune responses several cells and molecules participate in a complex set of events to induce antibody synthesis in B lymphocytes. Thus, antigen-presenting cells, specic T lymphocytes and dierent interleukins interact in dierent ways to activate specic B cells. But there are some antigens that directly activate B lymphocytes into antibody synthesis. These antigens are termed thymus-independent (TI) to indicate that they do not require helper T lymphocytes or any other help to induce a specic immune response. It has recently been shown that some TI antigens have the capacity to induce innate defence reactions in insects. Receptors that recognize these TI antigens (patternrecognizing receptors; PRRs) have been identied and cloned in mice and humans, and it is established that TI immune responses represent an evolutionary early and preserved defence reaction against various microorganisms in insects and mammals.
. Conclusion
eects, including toxicity and the ability to induce toxic shock. However, in nontoxic doses LPS induces a specic immune response in most animals and in most mouse strains. Immunogenicity is equal in normal mice and in strains lacking T cells, and LPS is the clearly dened TI antigen. LPS induces a specic immunoglobulin (Ig) M antibody response and there is no switch to other immunoglobulin classes. The immune response exhibits regular cycles (Figure 1). The mechanism of this cyclical appearance of antibodies to LPS (and also to other TI antigens of both type I and type II) is well studied and depends on the persistence of the antigen in the mouse in an immunogenic form for prolonged time periods, and on the
Antibody-forming cells per spleen x 103
Type I antigens
The classical TI type I antigen is lipopolysaccharide (LPS) from Gram-negative bacteria. LPS has many biological
Figure 1 Typical cyclical appearance of immunoglobulin (Ig) M antibody-producing cells in the response to either lipopolysaccharide (LPS) (thymus independent (TI) type I) or dextran (TI type II) after in vivo immunization. If a switch to IgG3 antibodies occurs, the second peak will not appear.
ability of antibodies to inhibit their own synthesis in a feedback regulatory system. Thus, LPS induces a primary anti-IgM response and, when the antibodies have reached a suciently high level, they will inhibit their own response, probably by binding to the antigenic determinants of LPS and preventing it from interacting with the B-cell immunoglobulin antigen receptor (BCR) on the B cells. Because IgM antibodies are rapidly catabolized in vivo, they will rapidly disappear and LPS will again be able to interact with the specic B cells and induce a second peak of antibody production. Experimental evidence for this mechanism will be presented later in connection with the immune response to the type II antigen dextran. There is no secondary immune response to TI type I antigens, indicating that T cells are required for the induction of B-cell memory.
Polyclonal B-cell activation by LPS LPS Polyclonally activating determinant (PAMP) Antigenic determinant
It was concluded that PBAs could activate B cells without binding to or otherwise interacting with their immunoglobulin receptors (Figure 2). Consequently, it was suggested that B cells expressed a new type of receptor, termed activation receptors, that was expressed by a large number of B cells and distinct from the Ig receptors. These receptors have now been found to be PRRs (see below). Later, several other PBAs were found and it was observed that all PBAs known were also TI antigens (Coutinho and Mo ller 1975a, Coutinho and Mo ller 1975b) (Table 1). However, the dose required for induction of polyclonal antibody synthesis was 100 to 1000 times higher than that required for induction of specic TI immune responses.
Table 1 B-lymphocyte activators Thymus-independent antigens LPS from Gram-negative bacteria Pneumococcal polysaccharide SIII Dextrans from Leuconostoc and levans from Pseudomonas Polymerized agellin from Salmonella Ficoll Polyvinylpyrrolidone (PVP) Polyclonal B-cell activators LPS from Gram-negative bacteria Pneumococcal polysaccharide SIII Dextrans from Leuconostoc and levans from Pseudomonas Polymerized agellin from Salmonella Ficoll Lipoprotein from Gram-negative bacteria Biostim from Klebsiella pneumoniae bacteria Mycoplasma Protein A from Staphylococcus aureus Puried protein derivative of tuberculin
LPS, lipopolysaccharide.
Figure 3 B cells are activated by polyclonal B-cell activators (PBAs), which do not interact with the BCRs (a). However, if the Ig receptors recognize antigenic determinants on the PBA, they will bind them with very high affinity. In this way a low concentration of a PBA can activate specific cells into specific antibody synthesis, although activation is (immunologically) nonspecifically delivered to pattern-recognizing receptors (PRRs) from pathogen-associated molecular patterns (PAMPs) on the PBA (b). LPS, lipopolysaccharide.
receptor. Such a B-cell subpopulation was very large, comprising about 30% of all B cells being activated by, for example, LPS. In contrast, a B-cell clone is dened as those B cells expressing identical BCRs. A B-cell clone is very small, typically only 0.0010.0001% of all B cells. It follows that, within each subpopulation of B cells, all B-cell clones were represented. The concept of B-cell subpopulations is illustrated in Figure 4. A large number of observations substantiated the subpopulation concept. Thus, during neonatal development the ability to become activated by dierent PBAs appeared at dierent times; genetic defects aecting the ability to become activated by a particular PBA (e.g. LPS) did not aect the ability of other PBAs to activate B cells (see below); some PBAs induced only antibody synthesis and not cell division, whereas others preferentially induced division and little antibody synthesis; and a third group, typically represented by LPS, induced both division and antibody synthesis. Direct evidence for the existence of dierent subpopulations responding to dierent PBAs was obtained from studies of immunological tolerance and the genetics of PBA activation.
The relationship between specic induction of TI immune responses and polyclonal activation is outlined in Figure 3. In short, the activation receptors were considered to have a very low binding anity for the activating determinants (now known to be pathogenassociated molecular patterns; PAMPs) of the PBA (or TI antigen). Therefore, very high doses of the polyclonal activators were required to obtain a sucient number of interactions between the activation receptor and the corresponding activation structure on the inducer. However, when the B cells could bind the TI antigen by its highanity BCRs, many fewer TI molecules were required to trigger the B cells into antibody synthesis. In the latter case the B cells only produced antibodies against the antigenic determinant present on the TI antigen (specic induction of a TI immune response). However, the antigenic determinants recognized by the BCRs were dierent from the activation receptors. When very high doses of TI antigens were added, B cells of all Ig specicities would be activated to antibody production, except those B cells specically recognizing the antigenic determinants: they were tolerized (see below).
B-cell Subpopulations
It was soon observed that all B cells could not be activated by any particular PBA or TI antigen (Gronowicz et al., 1974). The concept of B-cell subpopulations was introduced to dene B cells that possessed one type of activation
B-cell clones and subpopulations A B-cell subpopulation with the same activation receptors but different Ig receptors Activation receptor (PRR) 1 Activation receptor (PRR) 1 Activation receptor (PRR) 1 A B-cell clone with different activation receptors but identical Ig receptors Activation receptor (PRR) 2
BCR 1
BCR 2
BCR 1
BCR 1
Figure 4 A B-cell subpopulation has the same activation receptor (pattern-recognizing receptor; PRR) but different immunoglobulin (Ig) receptors. A subpopulation can be up to 30% of all B cells. A B-cell clone has identical Ig receptors, but different activation receptors. A clone is about 0.001 0.0001% of all B cells.
tolerogen by the addition of LPS in polyclonally activating concentrations (Mo ller et al., 1976). Apparently, B-cell tolerance did not aect all specic B cells within a clone; it was always possible to induce antibody synthesis against the tolerogen by the addition of a PBA. A detailed analysis of the mechanism of tolerance induction to the TI antigen dextran is illustrated in Figure 5. The immune response to dextran is very restricted and determined by genes in the Ig locus. The antibodies against dextran have the same idiotype and therefore belong to the same clone. Dextran cannot be catabolized in mice, because they lack the enzyme dextranase, but if this enzyme is given to mice previously injected with dextran there is complete removal of dextran from the mice.
Tolerance only affects part of the specific B-cell clone 4 PFC per spleen 103 4
Mice were tolerized against dextran by a high dose of the TI antigen dextran (Mo ller and Fernandez, 1978). Thereafter, dextran was removed from the system by treatment with dextranase. A short time period later the mice were immunized with dextran itself or by a thymus-dependent dextranprotein conjugate. As shown in Figure 4, the mice immunized with dextran remained unresponsive, but those immunized with the thymus-dependent dextranprotein conjugate showed a normal antidextran immune response. An interpretation of the experiments at the cellular level is illustrated in Figure 6. This shows that tolerance to the TI antigen dextran did not result in elimination of the entire clone of antidextranspecic B cells. Those antidextran-specic B cells that could be activated by helper T cells remained intact and could be induced to produce antidextran antibodies by a thymus-dependent form of dextran (or by LPS). It was concluded that only those B cells that possessed both activation receptors and BCRs to dextran were rendered tolerant (Figure 5). These ndings clearly illustrate that there exist B-cell subpopulations reactive with dierent Bcell activators and that, within one clone of specic Ig receptor-expressing B cells, there exist dierent subpopulations reactive with dierent B-lymphocyte activators.
Figure 5 Tolerance to the thymus-independent (TI) antigen dextran. Mice were left untreated or made tolerant to 10 mg dextran. After 4 days half of the mice were treated with dextranase (D:ase) to remove dextran and thereafter immunized with dextran itself (left) or a thymus-dependent (TD) dextran conjugate (right). Although the mice remained tolerant of dextran, they produced a normal antidextran immune response to the TD dextran protein conjugate.
Only B cells with Ig and activation receptors (PRRs) for the antigen can be tolerized
Cell death
T helper cell
Figure 6 Explanation at the cellular level of experiments shown in Figure 5. Two cells belonging to the same antidextran-specific clone are shown. In (a) the cells can bind dextran with its immunoglobulin (Ig) receptor and can react to the pathogen-associated molecular pattern (PAMP) of dextran with its pattern-recognizing receptor (PRR). As a very high dose of dextran was given, these cells will become tolerant and probably die. In (b) the cells also bind dextran with its Ig receptor, but remain unaffected because they possess PRRs that do not recognize dextran. When dextran has been removed by dextranase treatment, the cell can be activated by T helper cells, which secrete or possess PAMPs activating a corresponding PRR on the B cell. TI, thymus independent; TD, thymus dependent.
Meo, 1978), although F1 hybrid crosses indicated that the defective genes involved in the two strains were dierent. It was also found that B cells from C3H/HeJ mice could not be activated by LPS into polyclonal antibody synthesis (Coutinho et al., 1975a, Coutinho et al., 1975b and Watson and Riblet, 1974), although they were activated by a number of other PBAs. Furthermore, the mice did not produce antibodies to LPS or any hapten conjugated to LPS, although the same haptens conjugated to dierent carriers induced normal responses. It was concluded that the defect in this strain concerned activation receptors for LPS on B cells, which had a normal Ig repertoire. It was also clear that B cells from C3H/HeJ only lost responsiveness to LPS as a polyclonal activator and as a specic TI antigen, but had preserved responsiveness to all other PBAs tested and produced antibodies to all TI antigens, except to LPS and any antigenic determinant conjugated to LPS (Figure 6). Apparently the defect involved an activation receptor for LPS that was expressed only on some B cells (on one subpopulation of B cells). The genetic crosses that had been analysed (see list) suggested that in C3H/HeJ a defective receptor for LPS activation was expressed, but not functional, whereas the
defect in C57BL/10ScCr involved a loss mutation with no expression of the activation receptor. . C3H/HeJ mice are nonresponders to lipopolysaccharide . C57BL/10ScCr mice are also nonresponders . (C3H/HeJ C57BL/10ScCr) F1 hybrids are nonresponders (allelic genes) . (C3H/HeJ C3H) F1 hybrids are intermediate responders (co-dominance) . (C57BL/10ScCr C57BL) F1 hybrids are responders (dominant responsiveness) . The nonresponder gene (Lps) is on the fourth chromosome . The Lps mutation is in the Tlr4 gene . There is a point mutation (C3H/HeJ) or a loss mutation (C57BL/10ScCr) in this gene These conclusions were recently veried by molecular analysis of Lps genes in these two strains (Poltorak et al., 1998; Qureshi et al., 1999). It was found that the Lps gene belonged to the Toll family of genes (Tlr4) expressed in insects and mammals, and involved both in dorsoventral embryonic dierentiation in Drosophila and in innate defence against microbial infections in insects and
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mammals. In C3H/HeJ mice a point mutation had occurred that resulted in the expression of a defective gene product, whereas in C57BL/10ScCr mice a nonsense mutation resulted in lack of expression of the gene product (Qureshi et al., 1999), thus conrming the functional studies done previously.
to the Toll family, genes that are common to insects and mammals. These genes code for receptors used in innate defence against microorganisms in insects and presumably also in mammals, but the same genes are used in specic immune responses in mammals. In mammals they code for activation receptors expressed in B lymphocytes and activation results in the synthesis of immunoglobulins, rather than various defence molecules. There are several LPS receptors known in mammals. The LPS-binding proteins (LBPs) are found in serum and the CD14 molecules (binding LBPLPS complexes) are expressed on monocytes. As these molecules are normally expressed in the LPS mutant strains, they cannot be directly responsible for LPS unresponsiveness in these strains. In addition, CD14 is not expressed in B cells. It is clear therefore that the Lps (the Tlr4) gene product is directly responsible for the inability of B cells from C3H/ HeJ mice to respond polyclonally or specically to LPS or to any antigenic determinant conjugated to LPS. The LPSbinding protein and CD14 may function in monocytes/ macrophages to activate these cells after microbial infection, and the Tlr4 gene product may also function in these cells. However, in B lymphocytes only the Tlr4 gene product seems to be involved (Figure 7).
Normal mice LPS (PRR) receptor LPS (PRR) receptor No LPS (PRR) receptor
BCR 2
BCR 1
BCR 2
BCR 2
BCR 1
BCR 2
Figure 7 B lymphocytes in normal mice express both activation (pattern-recognizing receptor; PRR) and immunoglobulin (Ig) receptors. However, in lipopolysaccharide (LPS) mutant mice the activation receptor is not expressed (in C57BL/10ScCr mice), as shown in the figure, or else a defective product of the Lps (Tlr4) gene is expressed (in C3H/HeJ mice). TH, T helper cell.
same genes and their products are used in both systems. The PRRs are germline-encoded receptors that are expressed in B cells and monocytes in mammals. PRRs recognize invariant patterns on PAMPs, and dierent PRRs recognize dierent PAMPs. Activation receptors for TI antigens are germline encoded by genes belonging to the Toll family, and the rst identied mammalian gene (Lps or Tlr4) is critical for the induction of specic immune responses to LPS. The immune response to other TI antigens do not require the Lps genes, suggesting that there are several more genes to be found coding for other activation receptors (PRRs) on B cells, expressed in dierent B-cell subpopulations. These genes will probably be found to belong to the Toll family.
histocompatibility complex. In mammals, the expression of PRRs appears to be restricted to B lymphocytes. However, activated T lymphocytes can induce polyclonal antibody synthesis in B lymphocytes (Coutinho et al., 1984), suggesting that T cells possess PAMPs capable of activating B cells into antibody production. No PAMPs have yet been found among the various interleukins synthesized by activated T lymphocytes, but the elusive T-cell replacing factor (Schimpl and Wecker, 1972) or the CD40 ligand may be candidates for T-cell PAMPs. The corresponding PRR on B cells could be CD40.
Conclusion
Induction of TI immune responses requires the expression of activation receptors on B cells. These activation receptors are not the somatically encoded Ig receptors, but germline-encoded activation receptors expressed in a large proportion of B lymphocytes (approximately 30%), although not in all B cells. The rst gene identied in mammals that codes for one of these receptors is the Lps gene, identical to one member of the Toll family genes (Tlr4) which codes for defence molecules used in innate defence against various microorganisms in insects. Thus, gene products responsible for innate defence against
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pathogens in insects are used for the induction of specic immune responses in mammals. These PRRs in insects and mammals are used as activation receptors for induction of specic TI immunity in mammals, and the PAMPs found in groups of microorganisms exist in TI antigens and are responsible for activation of B cells into antibody synthesis.
References
Andersson J, Sjo berg O and Mo ller G (1972) Induction of immunoglobulin and antibody synthesis in vitro by lipopolysaccharide. European Journal of Immunology 2: 349353. Boman HG (1998) Gene-encoded peptide antibiotics and the concept of innate immunity: an update review. Scandinavian Journal of Immunology 48: 1525. Carrol MC and Janeway CA (1999) Innate immunity. Current Opinion in Immunology 11: 112. Coutinho A and Meo T (1978) Genetic basis for unresponsiveness to lipopolysaccharide in C57BL/10ScCr mice. Immunogenetics 7: 1724. Coutinho A and Mo ller G (1975a) Thymus-independent antigen B-cell induction and paralysis. Advances in Immunology 21: 114236. Coutinho A and Mo ller G (1975b) B cell mitogenic properties of thymusindependent antigens. Nature New Biology 245: 1214. Coutinho A, Gronowicz E and Sultzer B (1975a) Genetic control of Bcell responses. I. Selective unresponsiveness to lipopolysaccharide. Scandinavian Journal of Immunology 4: 139143. Coutinho A, Mo ller G, Gronowicz E (1975b) Genetical control of B-cell responses. IV. Inheritance of the unresponsiveness to lipopolysaccharides. Journal of Experimental Medicine 142: 253258. Coutinho A, Probor G, Pettersson T et al. (1984) T cell-dependent B cell activation. Immunological Reviews 78: 211224. Gronowicz E, Coutinho A and Mo ller G (1974) Dierentiation of B cells. Sequential appearance of responsiveness to polyclonal B cell activators. Scandinavian Journal of Immunology 3: 413421. Hammarstro m L, Smith CIE, Primi D and Mo ller G (1976) Induction of autoantibodies to red blood cells by polyclonal B cell activators. Nature 263: 6061. Kopp EB and Medzhitov R (1999) The Toll family and control of innate immunity. Current Opinion in Immunology 11: 1318. Mo ller G and Fernandez C (1978) Immunological tolerance to the thymus-independent antigen dextran can be abrogated by thymusdependent dextran conjugates. Evidence against clonal deletion as the mechanism of tolerance induction. Scandinavian Journal of Immunology 8: 2937.
Mo ller E and Sjo berg O (1972) Antigen-binding cells in immune and tolerant animals. Transplantation Review 8: 2649. Mo ller G, Gronowicz E, Persson U, Coutinho A and Mo ller E (1976) Spleen cells from animals tolerant to a thymus-dependent antigen can be activated by LPS to synthesize antibodies against the tolerogen. Journal of Experimental Medicine 143: 14291438. Mond JJ, Vos Q, Lees A, Snapper CM (1995) T cell independent antigens. Current Opinion in Immunology 7: 349354. Poltorak A, He X, Smirnova I et al. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282: 20852088. ` re L, Leveque G et al. (1999) Endotoxin-tolerant Qureshi ST, Larivie mice have mutations in Toll-like receptor 4 (Tlr4). Journal of Experimental Medicine 189: 615625. Schimpl A and Wecker E (1972) Replacement of T cell function by a T cell product. Nature 237: 1517. Sultzer BM (1968) Genetic control of leukocyte responses to endotoxin. Nature 219: 12531254. Ulevitch RJ and Tobias PS (1999) Recognition of Gram-negative bacteria and endotoxin by the innate immune system. Current Opinion in Immunology 11: 1927. Watson J and Riblet R (1974) Genetical control of responses to bacterial lipopolysaccharides in mice. I. Evidence for a single gene that inuences mitogenic and immunogenic responses to LPS. Journal of Experimental Medicine 140: 11471153. Watson J, Kelly K, Largen M and Taylor BA (1978) The genetic mapping of a defective LPS response gene in C3H/HeJ mice. Journal of Immunology 20: 422424.
Further Reading
Boman HG (1998) Gene-encoded peptide antibiotics and the concept of innate immunity: an update review. Scandinavian Journal of Immunology 48: 1525. Carrol MC and Janeway CA (1999) Innate immunity. Current Opinion in Immunology 11: 112. Coutinho A and Mo ller G (1974) Immune activation of B cells: evidence for one non-specic triggering signal not delivered by the Ig receptors. Scandinavian Journal of Immunology 3: 133146. Kopp EB and Medzhitov R (1999) The Toll family and control of innate immunity. Current Opinion in Immunology 11: 1318. Ulevitch RJ and Tobias PS (1999) Recognition of Gram-negative bacteria and endotoxin by the innate immune system. Current Opinion in Immunology 11: 1927.