Mice rendered deficient in lymphotoxin (LT) by gene targeting in embryonic stem cells have no morphologically detectable lymph nodes or Peyer's patches, although development of the thymus appears normal. Within the white pulp of the spleen, there is failure of normal segregation of B and T cells. Spleen and peripheral blood contain CD4+CD8- and CD4-CD8+ T cells in a normal ratio, and both T cells subsets have an apparently normal lytic function. Lymphocytes positive for immunoglobulin M are present in increased numbers in both the spleen and peripheral blood. These data suggest an essential role for LT in the normal development of peripheral lymphoid organs.

Background Endomyocardial biopsy is currently the standard method used to diagnose myocarditis. However, it is invasive and has a low diagnostic yield. Because the histological diagnosis of myocarditis requires the presence of myocyte injury, we sought to determine whether measurement of cardiac troponin I (cTnI), which is a serum marker with high sensitivity and specificity for cardiac myocyte injury, could aid in the diagnosis of myocarditis. Methods and Results To validate this approach, cTnI values were first measured in mice with autoimmune myocarditis. cTnI values were elevated in 24 of 26 mice with myocarditis but were not elevated in any of the control animals ( P <.001). Next, cTnI values were measured in the sera from 88 patients referred to the Myocarditis Treatment Trial and were compared with creatine kinase–MB (CK-MB) values measured in the same patients. cTnI values were elevated in 18 (34%) of 53 patients with myocarditis and in only 4 (11%) of 35 patients without myocarditis ( P =.01). In contrast, CK-MB values were elevated in only 3 (5.7%) of 53 patients with myocarditis and 0 of 35 patients without myocarditis ( P =.27). Thus, elevations of cTnI occurred more frequently than did elevations of CK-MB in patients with biopsy-proven myocarditis ( P =.001). Importantly, elevations of cTnI in patients with myocarditis were significantly correlated with ≤1 month duration of heart failure symptoms ( P =.02), suggesting that the majority of myocyte necrosis occurs early, and thus the window for diagnosis and treatment may be relatively brief. Conclusions cTnI was superior to CK-MB for detection of myocyte injury in myocarditis, and cTnI elevations were substantially more common in the first month after the onset of heart failure symptoms.

SummaryImmune interactions in the heart were studied using a murine model of myosin-induced autoimmune myocarditis. A T cell hybridoma specific for mouse cardiac myosin was generated from A/J mice and used to demonstrate that endogenous myosin/I-A k complexes are constitutively expressed on antigen-presenting cells in the heart. This T cell hybridoma, Seu.5, was used as a functional probe to identify a myocarditis-inducing epitope of cardiac myosin. Overlapping peptides based on the cardiac myosin heavy chain cx (myhcot) sequences were synthesized and tested for their ability to stimulate Seu.5 T cells. One peptide, myhco~(325-357) strongly stimulated the Seu.5 T cells, localizing the epitope to this region of the myhca molecule. Using truncated peptides, the epitope was further localized to residues 334-352. The myhccx(334-352) peptide strongly induced myocarditis when administered to A/J mice, which was histologically indistinguishable from that induced by myosin. The myhcot(334-352) epitope was present in cardiac myosin and not skeletal muscle myosins, providing a biochemical basis for the cardiac specificity of this autoimmune disease. Induction of myocarditis by this epitope was restricted to the myhcot isoform and not the myhc[3 isoform, suggesting there may be a difference in the efficiency of generating tolerance to these isoforms of cardiac myosin, which are differentially developmentally regulated. The myhcot(334-352) epitope bound to purified I-A k molecules in a similar manner to other I-Ak-restricted immunogenic epitopes, HEL(48-61) and R.Nase(43-56). Importantly, the myhc0~(334-352) epitope was able to bind to I-A k molecules on the surface of antigen-presenting cells in a stable manner. These findings demonstrate that autoantigenic epitopes can behave in a dominant manner and constitutively bind to class II molecules in the target organ in a similar manner to foreign immunogenic epitopes.

Determining how an autoimmune response is initiated is essential to understanding the mechanisms of autoimmunity. Self-reactive T cells, self-protein, and a failure of tolerance to that self-protein are all involved in the pathogenesis of autoimmune disease; yet it is not clear how self-reactive T cells rind the target self-protein to initiate an autoimmune response. Although a variety of self-proteins have been shown to be presented on both class I and class II major histocompatibility complex (MHC) molecules, the relationship of these self-proteins to autoimmune disease has not been established. To explore this further, we generated a T-cell hybridoma that recognizes mouse cardiac myosin, the self-protein that induces murine autoimmune myocarditis. Using this hybridoma as a probe to detect myosin-class II MHC complexes, we isolated a class II MHC+/CD45+ residential antigen-presenting cell (APC) population directly from the hearts of normal mice and looked for evidence ofendogenous processing ofcardiac myosin by these APC. In this report we show that myosin-class II MHC complexes are found on residential APC in the normal mouse heart. Induction of autoimmune myocarditis increased the expression of myosin-class H MHC in the heart and enhanced their APC functions. This result is a direct demonstration that epitopes of a self-antigen involved in initiating an autoimmune disease are endogenously processed and presented within the target organ.It is vital that the immune system distinguish between selfproteins and foreign protein antigens to elicit an effective immune response. Precisely how this discrimination is made is not completely understood, but negative selection (1-7), clonal anergy (8), peripheral tolerance (9), and downregulation of self-reactive T-cell receptors (10) are all mechanisms shown to function in vivo to sustain tolerance to self-proteins. Work by several investigators has firmly established that self-proteins are processed and presented by antigen-presenting cells (APC) (11)(12)(13)(14)(15). Wekerle et al. (16) reported that Schwann cells could process the self-protein myelin basic protein, known to initiate experimental allergic encephalomyelitis, and present it to myelin basic proteinreactive T cells, but this occurs only if the Schwann cells are first treated with interferon y in vitro. Recently, endogenously processed self-peptides have been eluted from both major histocompatibility complex (MHC) class I and class II molecules, sequenced, and identified (17-21); these selfpeptides represent a spectrum of nuclear, cytoplasmic, and membrane-bound self-proteins. Thus, it is clear that many self-proteins are endogenously processed and complexed with MHC class I or class II molecules. However, none ofthe currently identified self-proteins known to be endogenously processed by APC in vivo has been implicated in an autoimmune disease.Several models of autoimmune disease use a purified self-protein emulsified in complete Freund's adjuvant (CFA) to induce an autoimmune response in suscep...

Tumor necrosis factor (TNF) and interferon gamma (IFN-gamma) are pluripotent cytokines and have multiple functions during the inflammatory response. Using a murine model of autoimmune myocarditis, we studied the role of TNF and IFN-gamma in myocardial inflammation. Neutralizing monoclonal antibodies against TNF-alpha/beta and IFN-gamma were administered to myosin-immunized A/J mice to assess the effect on the severity of myocardial inflammation. Anti-TNF treatment significantly reduced the severity of myocarditis compared with rat immunoglobulin G or saline controls (p less than 0.0007) when given before myosin immunization. Myosin-specific lymph node T-cell proliferation studies showed no difference in the proliferative response between the anti-TNF-treated mice and controls. Administration of anti-TNF to mice after myosin immunization had no effect on the severity of inflammation. This suggests that TNF is an important mediator early in the pathogenesis of myocardial inflammation in this model of myocarditis. Neutralization of IFN-gamma significantly increased the severity of myocarditis compared with rat immunoglobulin G and saline controls (p less than 0.0065), suggesting that IFN-gamma may function as an important regulatory cytokine early in the pathogenesis of myocardial inflammation. Understanding the functions of cytokines during the inflammatory response to myocardial injury may provide important information on possible methods to limit myocardial damage.