The story begins with the production of a line of mice that are transgenic for a T-cell receptor (TCR) that was originally isolated from a T-cell clone specific for the bovine ribonuclease peptide residues 41-61, presented by the I-Ak MHC class II molecule. In the arcane lingo of immunologists, this TCR is said to be specific for ribonuclease 41-61 and restricted by the I-Ak molecule, resulting in the majority of T cells in the periphery expressing this TCR. In this transgenic mouse, however, the level of expression of the transgenic TCR in the periphery was low.
In order to introduce a TCR α-chain-null mutation, Benoist and Mathis crossed their TCR transgenic with a NOD line, which was available in the laboratory and which carried this targeted recombinant. Completely unexpectedly, mice expressing the TCR transgene and the I-Ag7 molecule developed a severe arthritis, with rapid onset beginning at about 3 weeks of age. The arthritis observed in these mice has several similarities to RA, including the following: the arthritis is chronic, progressive, symmetric, and exhibits a distal to proximal gradient of severity; it is MHC class II allele specific; and is characterized by synovitis, pannus formation, and cartilage and bone destruction, with increased production of TNF-α and interleukin-6, and partial dependence on the action of TNF-α. Additional characteristics that are unlike RA are the great severity, rapid progression, distal interphalangeal joint involvement, occasional mild involvement of the spine, and complete absence of rheumatoid factor activity.
Over the past 5 years, the group of Mathis and Benoist have executed an experimental tour de force, culminating in the two studies referred to above [8,9]. By a variety of crosses, including the introduction of an I-Ag7 β-chain transgene, they have demonstrated an absolute requirement for the presence of this MHC class II molecule for the development of the arthritic phenotype [10]. Additional crosses involving several types of targeted recombination (knockout) mice have also established an absolute requirement for both T cells (expressing the transgenic TCR) and B cells in the spontaneous development of the disease [10].
To explore this phenomenon further, the group of Benoist and Mathis [11] next carried out a series of experiments utilizing transfer of splenocytes, purified spleen CD4+ T cells, and purified B cells from arthritic donors into a variety of recipients, with and without the I-Ag7 molecule, and into a variety of recipients completely lacking their own T and B cells. These studies showed that an I-Ag7 restricted B-cell function, as well as the transgenic TCR, is critical for the development of the disease. The use of mouse lines expressing a restricted immunoglobulin repertoire (through the introduction of rearranged VDJ heavy chain and/or VJ light chain immunoglobulin gene segments 'knocked-in' to the germline) showed that, as the immunoglobulin repertoire became more and more restricted, arthritis development was delayed and, in the double heavy chain and light chain immunoglobulin transgenics, was completely eliminated. These results suggested that a specific B-cell product was required for the development of disease. Further studies (described in [11]) indicated that arthritis development required I-Ag7, and an I-Ag7 restricted, CD40-dependent, T cell-B cell interaction that occurred after the initial T cell stimulation. These results pointed to the possibility that immunoglobulins produced as a result of this interaction could be important effector molecules in the development of the arthritis.
Serum transfer studies [11] clearly established the ability of serum from arthritic, transgenic TCR- and I-Ag7-positive donors to induce the rapid onset of arthritis in both healthy and B-cell-deficient or lymphocyte-deficient recipients. Further experiments quickly established that the immunoglobulin G fraction of serum was capable of inducing arthritis in both normal and immunodeficient recipients. Serum-transferred arthritis was transient, resolving in 15–30 days, but persistent active arthritis could be induced by repeated injections of serum from arthritic donors. Additional transfers showed that the most severe arthritis developed in recipients that still expressed the transgenic TCR, showing that the specific T cells can increase the severity of the disease, even at late stages.
In the most recent studies [8,9] those investigators used serum from arthritic mice in immunoblots of tissue extracts from ankle, spleen, and kidney to show that this serum identified a 60-kDa protein that appears to be present in many different tissues. Purification of this 60-kDa protein, followed by trypsin digestion and amino acid sequencing of three of the tryptic peptides, revealed that all three peptide sequences were included in the amino acid sequence of a ubiquitous enzyme: GPI. The complementary DNA that encodes GPI was amplified by polymerase chain reaction, cloned, and placed in an Escherichia coli expression vector as a glutathione S-transferase (GST) fusion protein. This permitted the expression of GPI with the GST tag in E coli, and the construction of GPI-GST affinity columns, along with GST-only control columns. Using these columns, it was possible to show that all of the arthritogenic activity in arthritic donor serum was completely bound to the GPI-GST column, and that the flow-through immunoglobu-lins completely lacked the ability to transfer arthritis.
These experiments establish beyond any doubt that antibodies to GPI are the arthritogenic immunoglobulins that are contained in serum from the transgenic, arthritic mice. Further studies [9] showed that these antibodies are first detected in the blood at low concentrations at 3 weeks after birth — the time of arthritis onset — and increase steadily until age 8–10 weeks.
The authors then went on to show that the ribonuclease-specific, I-Ak-restricted, transgenic TCR is also specific for a peptide (as yet unidentified) from GPI when presented by I-Ag7. This situation is illustrated in Figure 1. Highly purified recombinant GPI presented by I-Ag7 antigen-presenting cells is capable of stimulating the transgenic Tcells to the same extent as the bovine ribonuclease peptide presented by I-Ak antigen-presenting cells [9].
Thus, this remarkable series of experiments has established that a disease primarily localized to joints can develop as a result of linked T-cell and B-cell autoreactivity for a self-antigen that is ubiquitously expressed in the cytoplasm of all cells in the body [9]. As the authors pointed out [9], arthritis does not occur as a result of other types of autoreactivity of this particular TCR. The same trans-genic TCR can also recognize an unknown self-peptide presented by an amino acid sequence variant of I-Ak. When this variant class II molecule is expressed in the TCR transgenic mice, however, there are no signs of joint pathology [8]. This result implies that there are highly specific properties of the recognition of a GPI peptide by the transgenic TCR. There may also be important and specific properties of the antigen GPI. This protein is expressed in the cytoplasm of all cells, and is also detected in the circulation. It is therefore possible that the rare B cell with an antibody specific for GPI may capture and present peptides from this molecule to T cells with a high degree of efficiency [9]. The antibody can then form complexes with the free GPI in the circulation, and this may be involved in the initiation of the arthritic process.
Much remains to be studied in this model before it is completely understood, and before the implications of this model for RA can be formulated. First, the identification of the specific peptide from GPI that is recognized by the transgenic TCR in the context of I-Ag7 must be determined. The affinity and the off-rate of this peptide in its interaction with I-Ag7 will undoubtedly be studied, as will the affinity and off-rate of the transgenic TCR for the specific peptide-I-Ag7 complex. Additional functional characterization of the cytokines produced by T cells that express this receptor in response to cognate peptide-MHC ligand, and the susceptibility of this cytokine pattern to manipulation also need to be analyzed in detail.
Numerous additional questions arise. Will immunization with GPI in either incomplete or complete Freund's adjuvant, with the induction of a diverse GPI-specific TCR repertoire, lead to the induction of arthritis and arthritic immunoglobulin G in I-Ag7 mice, or in mice with other MHC class II I-A alleles? These experiments would indirectly address several key questions concerning the nature of this autoantigen, and the nature and structure of the joint and the cells within the joint lining. Is there a unique property of this enzyme, or of other enzymes in the glycolytic pathway, that causes GPI and similar enzymes to localize to the joint? Does the subclass of the immunoglobulin G antibodies play a role in the development of the arthritis? Is this process dependent on expression of the stimulatory Fc receptors (FcRγ I and FcRγ III)? What is the role of complement in the arthritic process? Is there deposition of specific antibody, GPI, and components of the complement cascade?
Benoist and Mathis point out [8] that there may be similarities between this model and the findings referred to in a review by Zvaifler in 1973 [12]. It will be important to determine the localization of GPI, if any, in the normal joint and to enquire as to whether in the normal process of phagocytosis within the joint polymorphonuclear leuko-cytes extrude some of their intracellular enzymes during the course of phagocytosis within the joint, as suggested in the latter review [12]. Is the access of either antibody or immune complexes to the joint space greater than access to other tissue sites, or to endothelial sites that might also induce local inflammation? Zvaifler [12] cited a number of electron microscopic studies of synovial and joint tissues in RA, showing considerable deposition of immunoglobulin and complement. Are such deposits present in the GPI-specific arthritis model?
Benoist and Mathis [8] also noted several similarities between this model and the immune system-independent arthritis induced in mice by the expression of a human TNF transgene [13,14]. In this model, there is a high level of expression of human TNF in the synovial lining macrophages and fibroblasts. This finding underlines the importance of determining normal sites of expression of TNF in joint-related tissues, including synovial macrophages and subsynovial fibroblasts. As Benoist and Mathis pointed out [9], their model has many similarities to RA and is also dependent on TNF-α, in the sense that the severity of arthritis is much decreased in mice deficient in TNF receptor 1.
Two of the most important similarities between this model and RA are the demonstration that susceptibility to both diseases is MHC class II allele specific and the arthritic process is, to a great degree, dependent upon the action of TNF-α. The striking new finding in this model is the prominent role of specific antibody to a systemic, ubiquitously expressed cytoplasmic self-antigen. Production of this antibody is clearly T-cell-dependent, but once produced the antibody can transfer arthritis to naïve recipients (albeit the most severe form of the disease is dependent on the presence of specific Tcells and specific B cells producing a specific antibody).