The Specificity of Peptides Bound to Human Histocompatibility Leukocyte Antigen (HLA)-B27 Influences the Prevalence of Arthritis in HLA-B27 Transgenic Rats

Human histocompatibility leukocyte antigen B27 is highly associated with the rheumatic diseases termed spondyloarthropathies, but the mechanism is not known. B27 transgenic rats develop a spontaneous disease resembling the human spondyloarthropathies that includes arthritis and colitis. To investigate whether this disease requires the binding of specific peptides to B27, we made a minigene construct in which a peptide from influenza nucleoprotein, NP383-391 (SRYWAIRTR), which binds B27 with high affinity, is targeted directly to the ER by the signal peptide of the adenovirus E3/gp19 protein. Rats transgenic for this minigene, NP1, were made and bred with B27 rats. The production of the NP383-391 peptide in B27+NP1+ rats was confirmed immunologically and by mass spectrometry. The NP1 product displaced ∼90% of the 3H-Arg-labeled endogenous peptide fraction in B27+NP1+ spleen cells. Male B27+NP1+ rats had a significantly reduced prevalence of arthritis, compared with B27+NP− males or B27+ males with a control construct, NP2, whereas colitis was not significantly affected by the NP1 transgene. These findings support the hypothesis that B27-related arthritis requires binding of a specific peptide or set of peptides to B27, and they demonstrate a method for efficient transgenic targeting of peptides to the ER

Innate Immune Activation Can Trigger Experimental Spondyloarthritis in HLA-B27/Huβ2m Transgenic Rats

Spondyloarthritis (SpA) does not display the typical features of auto-immune disease. Despite the strong association with MHC class I, CD8+ T cells are not required for disease induction in the HLA-B27/Huβ2m transgenic rats. We used Lewis HLA-B27/Huβ2m transgenic rats [21-3 × 283-2]F1, HLA-B7/Huβ2m transgenic rats [120-4 × 283-2]F1, and wild-type rats to test our hypothesis that SpA may be primarily driven by the innate immune response. In vitro, splenocytes were stimulated with heat-inactivated Mycobacterium tuberculosis and cytokine expression and production was measured. In vivo, male and female rats were immunized with 30, 60, or 90 µg of heat-inactivated M. tuberculosis and clinically monitored for spondylitis and arthritis development. After validation of the model, we tested whether prophylactic and therapeutic TNF targeting affected spondylitis and arthritis. In vitro stimulation with heat-inactivated M. tuberculosis strongly induced gene expression of pro-inflammatory cytokines such as TNF, IL-6, IL-1α, and IL-1β, in the HLA-B27 transgenic rats compared with controls. In vivo immunization induced an increased spondylitis and arthritis incidence and an accelerated and synchronized onset of spondylitis and arthritis in HLA-B27 transgenic males and females. Moreover, immunization overcame the protective effect of orchiectomy. Prophylactic TNF targeting resulted in delayed spondylitis and arthritis development and reduced arthritis severity, whereas therapeutic TNF blockade did not affect spondylitis and arthritis severity. Collectively, these data indicate that innate immune activation plays a role in the initiation of HLA-B27-associated disease and allowed to establish a useful in vivo model to study the cellular and molecular mechanisms of disease initiation and progression

Relationship between inflammation, bone destruction, and osteoproliferation in the HLA-B27/human beta(2)-microglobulin-transgenic rat model of spondylarthritis

Objective Inhibition of inflammation and destruction, but not of osteoproliferation, in patients with spondylarthritis (SpA) treated with antitumor necrosis factor raises the question of how these three processes are interrelated. This study was undertaken to analyze this relationship in a rat model of SpA. Methods Histologic spine and joint samples from HLAB27/human beta 2-microglobulin (h beta 2m)transgenic rats were analyzed for signs of spondylitis and destructive arthritis and semiquantitatively scored as showing mild, moderate, or severe inflammation. Results In rats exhibiting spondylitis, mildly inflamed sections displayed lymphocyte infiltration in connective tissue adjacent to the junction of the anulus fibrosus and vertebral bone but not at the enthesis. Moderately inflamed tissue samples contained osteoclasts eroding bone outside the cartilage end plate. In sections from rats with severe inflammation, the cartilage end plate and underlying bone marrow were also affected. End-stage disease was characterized by complete destruction of the intervertebral disc and vertebrae, with ongoing infiltration. Osteoproliferation was not observed in samples from rats with no or mild inflammation, but was present at the edge of the vertebrae in sections with moderate inflammation and persisted during severe inflammation and end-stage destruction. Osteoproliferation occurred at the border of inflammation, at a distance from bone destruction. A strong correlation between the extent of inflammation, destruction, and osteoproliferation was observed. Sections from rats with arthritis displayed a similar pattern of synovial inflammation associated with bone destruction, and simultaneous but topographically distinct osteoproliferation starting from the periosteum. Conclusion SpA in B27/h beta 2mtransgenic rats is characterized by destructive inflammatory pannus tissue rather than by enthesitis or osteitis. Destruction and osteoproliferation occur simultaneously but at distinct sites in joints with moderate to severe inflammatio

Autoimmune epididymoorchitis is essential to the pathogenesis of male-specific spondylarthritis in HLA-B27-transgenic rats

Objective Male rats transgenic for HLAB27 and human beta 2-microglobulin (h beta 2m) spontaneously develop epididymoorchitis (EO) preceding the development of spondylarthritis (SpA). In the specific B27/h beta 2m-transgenic rat cross-strain (21-3 x 382-2)F1, only the males develop SpA, and neither sex develops gut inflammation. This study was undertaken to determine whether EO and SpA in male (21-3 x 382-2)F1 rats are causally related. In addition, the primary characteristics of EO in this rat arthritis model were assessed. Methods Male B27/h beta 2m-transgenic (21-3 x 382-2)F1 rats underwent bilateral, unilateral, or sham epididymoorchiectomy between ages 36 and 125 days. The castrated rats were given testosterone replacement. Alternatively, the 21-3 and 283-2 transgene loci were crossed with a transgene inducing aspermatogenesis. Rats were observed for the development of EO, arthritis, and spondylitis. Results In unmanipulated transgenic rats, inflammation was first evident in the ductuli efferentes (DE; ducts linking the rete testis to epididymis) as early as age 30 days. The inflammation was initially neutrophilic, and later became granulomatous. Antisperm and antitestis cell antibodies appeared in the rat serum after age 70 days. Cells infiltrating the testes were predominantly CD4+ T cells and CD68+ or CD163+ macrophages. Quantitative polymerase chain reaction of the DE, epididymis, and testis showed elevations in the levels of interferon-gamma, interleukin-10 (IL-10), and IL-17A. In addition, levels of IL-12A, IL-22, IL-23A, and IL-23 receptor were found to be elevated in the DE. Remarkably, castration of the rats before age 91 days completely prevented the subsequent onset of arthritis and spondylitis, as did transgene-induced azospermia. Conclusion Autoimmune EO develops spontaneously in HLAB27/h beta 2mtransgenic (21-3 x 283-2)F1 rats at age 30 days, the age when antigen-positive meiotic germ cells first exit the testis. Persistent testicular inflammation and/or antigenic stimulation are essential prerequisites for the subsequent development of SpA. Thus, dysregulated innate immunity at immune-privileged sites may be an essential mechanism triggering the onset of Sp