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Atherosclerosis is a multifactorial chronic inflammatory disease characterized by the presence of T-cells, macrophages, and dendritic cells in the arterial intima. Classical risk factors lead to over-expression of stress proteins, especially heat shock protein 60 (HSP60). HSP60 on the surface of arterial endothelial cells (ECs) then becomes a target for pre-existing adaptive anti-HSP60 immunity resulting in infiltration of the intima by mononuclear cells. In the present study, T-cells derived from early, clinically still inapparent human atherosclerotic lesions were analyzed phenotypically and for their reactivity against HSP60 and HSP60-derived peptides. HSP60 was detected in ECs and CD40- and HLA Class II-positive cells within the intima. Effector memory CD4(+) T-cells producing high amounts of interferon-γ and low levels of interleukin-4 were the dominant subpopulation. T-cells derived from late lesions displayed a more restricted T-cell receptor repertoire to HSP60-derived peptides than those isolated from early lesions. Increased levels of soluble HSP60 and circulating anti-human HSP60 autoantibodies were found in donors with late but not early lesions. This is the first functional study of T-cells derived from early human atherosclerotic lesions that supports the previously proposed concept that HSP60-reactive T-cells initiate atherosclerosis by recognition of atherogenic HSP60 epitopes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Heat shock protein 60 (HSP60), expressed on the surface of endothelial cells (ECs) stressed by e.g. oxidized LDL or mechanical shear, was shown to function as an auto-antigen and thus as a pro-atherosclerotic molecule. The aim of this study was to determine whether cigarette smoke chemicals can lead to the activation of the "HSP60 pathway." It was also our aim to elucidate the dynamics of HSP60 from gene expression to endothelial surface expression and secretion. Here we show for the first time that the exposure of human umbilical vein endothelial cells (HUVECs) to cigarette smoke extract (CSE) results in an up-regulation of HSP60 mRNA. Live cell imaging analysis of a HSP60-EYFP fusion protein construct transfected into ECs revealed that mitochondrial structures collapse in response to CSE exposure. As a result, HSP60 is released from the mitochondria, transported to the cell surface, and released into the cell culture supernatant. Analysis of HSP60 in the sera of healthy young individuals exposed to secondhand smoke revealed significantly elevated levels of HSP60. Cigarette smoking is one of the most relevant risk factors for atherosclerosis. Herein, we provide evidence that cigarette smoke may initiate atherosclerosis in the sense of the "auto-immune hypothesis of atherosclerosis.", (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Oxidative stress, continuously exerted during chronic inflammation, has been implicated as a major causative agent of cellular dysfunction and cell death. In the present study, we investigated the impact of oxidative stress on the mode of cell death in HUVECs using H2O2 as a model reagent. We found that the predominant form of cell death was necrosis. Necrosis induction was accompanied by a distinct mode of caspase-3 cleavage, yielding a 29-kDa fragment. While inhibition of caspases could not prevent the generation of the 29-kDa fragment, general protease inhibitors, such as leupeptin and LLNL, proved to be effective in inhibiting the distinct processing pattern of caspase-3. These results suggest that caspases can act as substrates for non-caspase proteases in cells primed for necrosis induction. Thus, the pattern of caspase-3 cleavage might reflect the proteolytic system engaged in the cell death machinery in HUVECs.

Autoimmune diseases in human patients only become clinically manifest when the disease process has developed to a stage where functional compensation by the afflicted organ or system is not possible anymore. In order to understand the initial etiologic and pathogenic events that are generally not yet accessible in humans, appropriate animal models are required. In this respect, spontaneously developing models--albeit rare--reflect the situation in humans much more closely than experimentally induced models, including knockout and transgenic mice. The present chapter describes three spontaneous chicken models for human autoimmune diseases, the Obese strain (OS) with a Hashimoto-like autoimmune thyroiditis, the University of California at Davis lines 200 and 206 (UCD-200 and -206) with a scleroderma-like disease, and the amelanotic Smyth line with a vitiligo-like syndrome (SLV). Special emphasis is given to the new opportunities to unravel the genetic basis of these diseases in view of the recently completed sequencing of the chicken genome.

During the past 30 years, much debate has centered around side effects of silicone breast implants. Meta-analyses rejected the presumed relationship between silicone breast implants and connective tissues diseases but, in seeming contradiction, case reports about connective tissue diseases and rheumatoid symptoms continue to be published. We analyzed the cellular and molecular composition of fibrous capsules removed from patients at various times after surgery for diagnostic purposes (breast cancer relapse) or to relieve painful constrictive fibrosis. Frozen sections of capsule tissue were immunohistochemically stained for subsets of lymphocytes, macrophages, dendritic cells, fibroblasts, smooth muscle cells, for collagenous and non-collagenous extracellular matrix proteins, for heat shock protein 60 (HSP60) and for adhesion molecules. Massive deposition of fibronectin and tenascin was observed adjacent to the implant surface. The capsule/silicone implant contact zone was consistently characterized by a palisade-like single or multilayered cell accumulation consisting of HSP60+ macrophages and HSP60+ fibroblasts. Mononuclear cell infiltrates consisting of activated CD4+ T-cells, expressing CD25 and CD45RO, as well as macrophages were detected beneath the contact zone as well as perivascularly. Importantly, many Langerhans-cell like dendritic cells (DCs) were found with a predilection at the frontier layer zone abutting the silicone implant. Also, at this site, massive expression of ICAM-1, but not VCAM-1 or ELAM-1 emerged. Endothelial cells of the intracapsular neovasculature were P-Selectin+. Our results show that silicone induces a strong local T-cell immune response and future studies will determine the specificity and function of these T-lymphocytes., (Copyright 2004 Elsevier Ltd.)

Systemic Sclerosis (SSc) is a connective tissue disease affecting the skin and internal organs. Retinopathy has been described in patients with SSc, but cannot be distinguished from secondary changes due to concomitant hypertension. UCD 200 chickens, a well-established animal model for SSc, were used in this study to investigate the posterior ocular segment for manifestations of SSc. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling was applied to detect endothelial cell (EC) apoptosis, a condition previously shown to represent the first step in SSc pathogenesis in humans and to be present in the skin and the involved internal organs of UCD 200 chickens in the acute stage. Our study showed a complete absence of EC apoptosis in the pecten and choroidal vessels of UCD 200 chickens in the acute stage. Ophthalmoscopy, biomicroscopy and histology revealed normal structures of the pecten, retina and choroid in the chronic stage. In summary, we showed that there is no primary involvement of the posterior ocular segment in avian SSc. SSc of UCD 200 chickens closely mimics human SSc, presenting all the clinical, serological and histological disease manifestations seen in the human counterpart. Therefore, our data raise serious doubts about primary posterior ocular involvement in human SSc. However, fundal examinations in patients with SSc may have their justification for assessment of hypertensive retinopathy.

The immune system interacts with the hypothalamo-pituitary-adrenal axis via so-called glucocorticoid increasing factors, which are produced by the immune system during immune reactions, causing an elevation of systemic glucocorticoid levels that contribute to preservation of the immune reactions specificities. Previous results from our laboratory had already shown an altered immuno-neuroendocrine dialogue via the hypothalamo-pituitary-adrenal axis in autoimmune disease-prone chicken and mouse strains. In the present study, we further investigated the altered glucocorticoid response via the hypothalamo-pituitary-adrenal axis in murine lupus. We established the circadian rhythms of corticosterone, dehydroepiandrosterone-sulfate, adrenocorticotropic hormone and melatonin, as well as the time response curves after injection of interleukin-1 of the first three parameters in normal SWISS and lupus-prone MRL/MP-fas(Ipr) mice. The results show that lupus-prone MRL/ MP-fas(Ipr) mice do not react appropriately to changes of the light/dark cycle, circadian melatonin rhythms seem to uncouple from the light/dark cycle, and plasma corticosterone levels are elevated during the resting phase. Diurnal changes of dehydroepiandrosterone-sulfate and adrenocorticotropic hormone were normal compared to healthy controls. These data indicate that MRL/ MP-fas(Ipr) mice not only show an altered glucocorticoid response mediated via the hypothalamo pituitary adrenal axis to IL-1, but are also affected by disturbances of corticosterone and melatonin circadian rhythms. Our findings may have implications for intrathymic T cell development and the emergence of autoimmune disease.

Systemic sclerosis (SSc) is a multisystem disorder characterized by mononuclear cell infiltration and fibrosis. Using skin samples from human SSc and UCD 200 chickens, which spontaneously develop a hereditary disease closely resembling human SSc, we have shown previously that endothelial cell apoptosis is a primary event in the pathogenesis of SSc. The aim of the present study was to investigate the initial disease stage in visceral organs of UCD 200 chickens with special emphasis on endothelial apoptosis, mononuclear cell infiltration and collagen deposition using tissue samples from oesophagus, lung, heart, kidney and liver. Apoptotic endothelial cells were detected by terminal deoxynucleotidyl transferase-mediated FITC-dUTP nick end labeling (TUNEL), mononuclear cell infiltrates were stained with hematoxylin and eosin, and increased collagen deposition was demonstrated by Goldner staining. Apoptotic endothelial cells were detected in oesophagus, lung and kidney of UCD 200 chickens at the initial stage of the disease. No apoptotic endothelial cells were found in heart or liver of UCD 200 or in visceral organs of healthy normal UCD 058 control chickens. Oesophagus of UCD 200 chickens, which was the most affected internal organ, showed mononuclear cell infiltrations and increased deposition of collagen. Perivascular inflammatory infiltrates and collagen deposition appeared later than endothelial cell apoptosis. These data support the hypothesis that endothelial cell apoptosis initiates the disease process, followed by mononuclear cell infiltration and fibrosis., (Copyright 2000 Academic Press.)

The involvement of CD4+ and CD8+ T cells in pathogenesis of spontaneous autoimmune thyroiditis (SAT) in obese strain (OS) chickens has not been studied in depth until now. We depleted CD4+ or CD8+ T cells in OS chickens by treatment with murine monoclonal anti-CD4 or anti-CD8 antibodies at 3 day intervals beginning at hatching. The birds were killed at 19-25 days of age. Treatment with anti-CD4 antibody completely prevented SAT development, while treatment with anti-CD8 antibody partially inhibited SAT. These results show the critical role of CD4+ T cells in the development of SAT in OS chickens, and indicate that CD8+ T cells are also involved in SAT pathogenesis.

University of California at Davis (UCD) lines 200 and 206 chickens develop a hereditary systemic scleroderma-like connective tissue disease characterized by severe lymphocytic infiltration and excessive accumulation of collagen in skin and internal organs. The immune system seems to play an important role in the development and/or perpetuation of this condition. The main goal of our work with this strain is the investigation of interactions between endothelial cells, lymphocytes, macrophages and fibroblasts leading to the proliferation of the latter and to excessive collagen synthesis and/or deposition. One aim of the present study was to clarify whether UCD-200 and 206 chickens have a defect of collagen genes at the genomic level by means of restriction fragment length polymorphism (RFLP) analysis using non-radioactively labelled cDNA probes specific for chicken alpha 1(I), alpha 2(I), alpha 1(II), alpha 1(III), alpha 1(VI), alpha 2(VI), and alpha 3(VI) (pro) collagens. As in the human disease, no gross alteration at the genomic level of collagen genes has been found, thus providing the UCD-200/206 model to be appropriate for studying the altered collagen metabolism in systemic sclerosis (SSc). In addition to the RFLP analysis of procollagen genes, we investigated the endogenous avian leukosis virus loci (ev) of UCD-200 and 206 chickens by means of Southern blot analysis of Sac I and BamH I digested DNA samples using pRAV-2, a Rous sarcoma virus specific probe, for hybridization. Most UCD-200 and 206 chickens harbour evs 1, 3 and 10 similar to the healthy control UCD-058, but they also contain a novel ev characterized by a 4.2 kb Sac I fragment and a 6.1 kb BamH I fragment, which we would like to designate ev 23. So far, the role of ev 23 in the development of avian scleroderma is unclear; for further analysis classical crossbreeding experiments are necessary and are underway.

We studied T cell receptor variable beta (TCR V beta) gene usage by autoreactive T cells in spontaneous autoimmune thyroiditis (SAT) of obese strain (OS) chickens. Chicken alpha beta T cells may express either V beta 1 or V beta 2 genes, the products of which can be recognized by TCR2 and TCR3 monoclonal antibodies, respectively. Selective depletion of V beta 1+ or V beta 2+ T cells in OS chickens was accomplished by repeated injections of TCR2 or TCR3 antibodies into embryonic and 1-3-week-old chickens. The birds were killed at 20 days of age and their spleens and thyroid glands evaluated by immunohistochemistry. We found that V beta 1+ T cells preferentially infiltrated OS chicken thyroid glands. Antibody treatments resulted in a 41% reduction in frequency of V beta 1+, and a 87% reduction of the frequency of V beta 2+ cells in the circulation, and in a profound decrease of the respective T cells in spleens and thyroid glands. Selective suppression of V beta 1+ T cells partially inhibited SAT development in that thyroid-infiltrating cells and destruction of thyroid follicles were reduced by more than 50%. Thyroglobulin autoantibody serum levels were also reduced in V beta 1+ T cell-depleted OS chickens, whereas selective depletion of V beta 2+ T cells did not inhibit SAT development. These findings indicate preferential TCR V beta 1 gene usage by autoreactive T cells in SAT of OS chickens.

MRL/Mp-lpr/lpr (MRL/lpr) mice carry a mutation in the Fas gene whose product is involved in the regulation of lymphocyte apoptosis. This mutation is associated with the lpr phenomenon, i.e., a massive expansion of phenotypically abnormal CD4-CD8- cells ("double negative," DN) alpha/beta T cells (lpr cells) that becomes manifest at 3-4 months of age. As in normal mice, intravenous SEB injection into 2- or 6-month-old female MRL/lpr mice causes a transient expansion of SEB-reactive V beta 8+ T cells, followed by a deletion of this subset. In contrast, in the same animals, the frequency of abnormal V beta 8+CD4-CD8- cells is not modulated by SEB. Whereas DN T cells are completely resistant to SEB-mediated deletion in vivo, their precursors appear susceptible to SEB-induced deletion. Thus, a single injection of SEB prior to the surge of DN T cells in peripheral lymphoid organs, at 2 months of age, is sufficient to cause a stable long-term (6 months) deletion of DN cells. This is accompanied by a significant amelioration of autoimmune parameters (autoantibody titers, incidence of arthritis and nephritis), thus pointing to the feasibility of employing superantigens for simple manipulations of the immune repertoire that result in the long-term prophylaxis of autoimmune diseases.

Considerable controversy exists concerning the role of aberrant MHC-class II antigen expression in the pathogenesis of organ-specific auto-immune disease. Since Obese strain (OS) chickens are afflicted with a spontaneously occurring autoimmune thyroiditis (SAT), we have readdressed this pivotal question by investigating the chronical appearance of MHC-class II antigens on thyroid epithelial cells (TEC) of OS and normal healthy CB chickens before onset of overt clinical symptoms in the former. Among the candidates as potent inducers of aberrant MHC-class II antigen expression, interest in our studies focussed on the potential role of viruses in the development of SAT. Since aberrant MHC-class II antigen expression could prove to be an epiphenomenon of virally afflicted TEC, we determined 2,5-oligoadenylate synthetase and 2,5-oligoadenylatepolymer cytosol levels in both chicken lines. Our results indicate that the presence of infiltrating lymphocytes does not necessarily represent a prerequisite for the aberrant expression of MHC-class II antigens but coincides in most cases. However, the phenomenon seems to play a perpetuating rather than a causative role. Moreover, in support of a possible viral involvement, elevated levels of the 2,5-oligoadenylate synthetase and 2,5-oligomers could be demonstrated in TEC cytosol of OS chickens.

We previously reported on an altered immune-endocrine feedback loop via the hypothalamo-pituitary-adrenal (HPA) axis in Obese strain (OS) chickens afflicted with spontaneous autoimmune thyroiditis. These animals are deficient in plasma corticosterone increase after antigenic challenge or application of cytokine-containing conditioned medium of mitogen-stimulated spleen cells (CM). To investigate whether the impaired ability to respond to cytokines with glucocorticoid-increasing factor (GIF) activity, e.g. interleukin 1 (IL 1), is restricted to OS chickens as a model for an organ-specific autoimmune disease, we extended our experiments to another autoimmune-prone animal strain, the chickens of the University of California at Davis line 200 (UCD-200). These animals develop an inherited inflammatory fibrotic disease that closely resembles human progressive systemic sclerosis (scleroderma). Application of GIF-containing CM to UCD-200 chickens leads to a transient increase in glucocorticoid serum levels within 1-2 hours comparable to that of controls. But, while corticosterone levels in the latter returned to normal baseline levels after 4 hours, they were still elevated in autoimmune chickens. Although the peak of the glucocorticoid hormone serum concentrations was equal to that of controls, UCD-200 had to secrete twice as much adrenocorticotropic hormone to achieve this corticosterone serum level due to an apparent hyporesponsiveness of the adrenal gland to this secretagogue. The altered cytokine-induced glucocorticoid secretion is found in early as well as in chronic, sclerotic stages of the disease. Cellular alterations in the peripheral blood of UCD-200 chickens during the prolonged elevated corticosterone section, i.e. between 2-4 hours after CM application, are characterized by a significant decrease in the percentage of CD4+ and CD8+ cells. Furthermore, a significant increase in B cells up to 24 hours with a maximum after 1 hour was found. The proliferative response to the mitogen concanavalin A of peripheral mononuclear cells was inversely correlated to the serum corticosterone level, showing a permanent decrease of 80-90% after 1-4 hours in autoimmune animals. This functional alteration in UCD-200 was accompanied by an 80% decrease in serum interleukin 2 (sIL 2) activity 4 hours after CM application. Twenty-four hours later an eight-fold increase in sIL 2 rebound activity was found, indicating that the inhibitory effect of corticosterone in UCD-200 chickens is not long-lasting.

An altered immunoendocrine feedback regulation within the hypothalamo-pituitary-adrenal axis may modulate the pathogenesis of an avian autoimmune disease. To date studies have been hampered by a lack of reliable, specific, and sensitive methods for determining adrenocorticotropic hormone (ACTH) in chickens. The present study describes the determination of ACTH in plasma of chickens with a commercial radioimmunoassay, the antibody of which binds to the midregion of human ACTH 1-39. The chickens, kept on a 12-hr day and 12-hr night shift with artificial light, showed changes in plasma ACTH concentrations during the light phase with maximum values 8 hr after the light was turned on. ACTH was not measurable after treatment with dexamethasone. Intravenous administration of supernatants from concanavalin A-stimulated spleen cells increased basal plasma ACTH concentrations more than 20-fold within 1 hr. This increase in plasma ACTH was higher and longer lasting in UCD 200 chickens, an animal model for scleroderma, compared with outbred and inbred normal White Leghorn chickens.

University of California, Davis, line 200 and 206 chickens spontaneously develop an autoimmune syndrome that has many features analogous to human scleroderma, including dermal fibrosis, antinuclear antibodies and antibodies to type II collagen. These birds also have thymic subcapsular epithelial defects and an abnormality in T cell calcium influx and proliferation in response to both T cell receptor-dependent and -independent activators. To determine whether fibroblast activation is a contributing factor to development of skin fibrosis in line 200/206 chickens, as it is in human scleroderma, we studied the collagen, non-collagenous protein and glycosaminoglycan (GAG) production of 34 separate fibroblast lines derived from the normal and fibrotic skin of line 200 and 206 chickens and from the skin of control chicken lines 058 and 254. The mean +/- SEM 24-h incorporation of 3H-proline or 3H-glucosamine into extracellular collagen, non-collagenous protein or GAG by first passage fibroblast lines derived from the fibrotic skin of diseased birds was 1,526 +/- 136, 859 +/- 82 and 25.7 +/- 1.3 dpm/10(3) cells, respectively, while fibroblast lines derived from the skin of control birds produced only 341 +/- 36, 343 +/- 42 and 15.2 +/- 1.4 dpm/10(3) cells. Similar differences in results were recorded for cell-associated production, and when collagen and non-collagenous protein production were assessed using non-radioactive electrophoretic methods. The activated phenotype of the fibroblast lines derived from the fibrotic skin of diseased birds persisted through 10 cell doublings in tissue culture. However, the ratio of type I:III collagen and the profile of GAG types produced were similar in all fibroblast lines studied. These results suggest that fibroblast activation is responsible for the skin fibrosis observed in this avian model of scleroderma.

T cell activation is dependent upon calcium influx and protein kinase C activation, with subsequent lymphocyte proliferation dependent upon IL-2. Abnormalities in T cell proliferation, including abnormal calcium influx and defective protein kinase C activation, have been identified in aged mice and humans and many autoimmune diseases including diabetes, lupus and scleroderma. Since UCD line 200 chickens, which spontaneously develop a scleroderma-like disease, have both thymic defects and a diminished peripheral blood lymphocyte response to IL-2, we have further investigated T cell function in these birds. Interestingly, line 200 T cells respond poorly in vitro to a variety of diversely acting T cell mitogens including concanavalin A, phytohemagglutinin and anti-chicken CD3 monoclonal antibody. Moreover, they do not respond well even to phorbol myristate acetate in conjunction with ionomycin. Addition of exogenous IL-2-containing supernatant concurrently with mitogenic stimulation also had no significant effect. Analysis of intracellular free calcium demonstrated that the lymphocytes from diseased birds had a reduced influx of calcium (or release for intracellular stores) following stimulation. These data clearly reflect a unique defect in T cell activation associated with avian scleroderma. Analysis of chicken CD3, CD4 and CD8 expression revealed a 39% decrease in peripheral blood CD4+ cells in scleroderma birds, although this decrease was not sufficient to explain the 80-90% decrease observed in proliferation assays and calcium influx. Our data support the hypothesis that avian scleroderma is mediated via abnormal function of lymphocyte co-stimulatory molecules or intracellular calcium regulators.

University of California at Davis line 200 (UCD-200) chickens develop a hereditary connective tissue disease characterized by severe lymphocytic infiltration, vascular occlusion and fibrosis of skin and internal organs. To identify cellular immunological abnormalities in the acute inflammatory disease stage of this animal model for progressive systemic sclerosis (PSS) we investigated the phenotypic characteristics and function of peripheral blood lymphocytes (PBL), spleen cells and thymocytes in comparison with skin infiltrating cells. Immunofluorescence and immunohistochemical analysis using monoclonal antibodies revealed the overwhelming majority of skin infiltrating mononuclear cells in the deeper dermis and subcutaneous tissue to be T cell receptor alpha/beta (TcR2)+/CD3+/CD4+/class II+ cells, a small portion (5-10%) of which were interleukin 2 (IL-2) receptor positive. In contrast, the inflammatory infiltrate in perivascular areas of the papillary dermis was constituted of mainly TcR gamma/delta (TcR1)+/class II- lymphocytes. Only few B cells (T/B cell ratio greater than 5) were detected. These diseased chickens showed significantly reduced percentages and numbers of circulating peripheral T cells exhibiting TcR1, TcR2, CD3, CD4 or IL-2-receptor, probably owing to an increased influx into lymphoid organs and affected tissues. In contrast to healthy chickens, the thymi of UCD-200 animals revealed fewer cells expressing TcR1, TcR2 and class II antigen, suggesting an altered intrathymic maturation of the T cell lineage. Functional in vitro studies showed a significantly decreased T cell mitogen-induced proliferation rate associated with a decreased capacity to produce IL-2 and to express IL-2 receptors. In contrast to the deficient in vitro IL-2 production the sera of UCD-200 chickens contained significant levels of IL-2 bioactivity. The alteration of T lymphocyte physiology in UCD-200 chickens adds, at least in part, to the parallels between this animal model and its human counterpart. These data confirm our hypothesis that the PSS-like disease of UCD-200 chickens includes a numeric and/or functional alteration of peripheral T cell subsets, especially of TcR1 positive cells, in contrast to the pronounced accumulation in the afflicted tissues.

There is evidence that fatty streaks in arteries can transform into atherosclerotic plaques. Mononuclear cells, including both monocytes and lymphocytes, are among the first cells participating in the development of atherosclerosis of experimental animals. To investigate the roles of different cell types in human atherosclerosis, we enumerated and compared the cellular compositions of normal intima, the transition zone (the area between the normal intima and the core of fatty streaks), fatty streaks, and plaques in young (age 16-30 years) and aged (over 60 years) human specimens using double-staining immunofluorescence with a series of monoclonal and polyclonal antibodies. T lymphocytes, both T helper/inducer (70% of T cells) and T suppressor/cytotoxic (30%) phenotypes, were found in every stage of atherosclerosis, constituting 30 to 40% of total cells in fatty streaks and transition zones of young subjects, and occasionally even in normal intima. Seventy percent of these T cells were HLA-DR positive, which indicated that most of them were activated. Macrophages were most frequent in fatty streaks and around the necrotic core of plaques. Smooth muscle cells, increasing from 5 to 30% with lesion progression, were HLA-DR positive where activated T helper cells occurred in the vicinity. The intracellular presence of the invariant gamma chain confirmed that HLA-DR was actually synthesized by these smooth muscle cells. Endothelial cells were HLA-DR positive above those regions of the lesions where HLA-DR-positive cells had accumulated, but not in normal intima, again suggesting induction of HLA-DR expression by T-cell-derived gamma-interferon. Furthermore, most HLA-DR-positive cells were also identified as HLA-DP and HLA-DQ positive. This aberrant major histocompatibility complex class II antigen expression in smooth muscle and endothelial cells may participate in the perpetuation of the atherogenetic autoimmune reaction.

We have recently reported a two-fold defect in glucocorticoid mediated immunoregulation in the Obese strain (OS) of chickens with spontaneous autoimmune thyroiditis (SAT): (i) a decreased basal corticosterone (CN) tonus due to an elevation of plasma corticosteroid-binding globulin (CBG) and (ii) an impaired CN rise in response to antigenic stimuli as well as lymphokines produced after mitogenic stimulation. The aim of the present study was to investigate the pathophysiological relevance of testosterone for the development of SAT. Compared to healthy normal White Leghorn chickens (NWL) the basal sex hormone tonus as well as androgen receptors of bursal tissue are not altered in the OS. Administration of sheep red blood cells (SRBC) or lymphokine containing conditioned media not only increased CN plasma levels but concomitantly modulated testosterone serum concentrations, although in an inverse direction and without significant difference between OS and healthy control chickens. These results suggest that, in contrast to the glucocorticoid system, androgen tonus as well as its modulation by immune signals are normal in the OS. The mode of action by which androgens exert their known beneficial effect on the development of SAT was also studied. According to our findings the capacity of testosterone to prevent SAT when administered during the posthatching period can be attributed to direct effects on bursal epithelial cells as well as indirect mechanisms, namely a fall in CBG levels leading to normalization of the CN tonus.

In this study we investigated the genetic background of primary abnormalities found in the thyroid gland of Obese strain (OS) chickens with spontaneous autoimmune thyroiditis (SAT), i.e., susceptibility to passively transferred antibodies to thyroglobulin (TgAb) and incomplete suppression of iodine uptake by thyroxine (T4). Several crosses between the B15/B15 subline of OS chickens and the inbred CB line (B12/B12) were done and the progeny was analyzed for thyroiditis after injection of OS serum containing high titers of TgAb. It was found that passive transfer of TgAb increased the lymphoid infiltration in the thyroids of OS chickens, but had no effect on CB birds. A genetic analysis of backcrosses revealed that this trait is, in the case of simple Mendelian inheritance, encoded by at least three recessive genes. The thyroidal 131I uptake of these crosses under T4 was also determined and we found that this trait is most probably encoded by only one recessive gene.

Aberrant expression of HLA-DR antigens on epithelial cells is seen in various organ-specific autoimmune disorders including Hashimoto thyroiditis (HT). Expression of HLA-DQ has so far not been demonstrated on these cells. We report here that thyroid epithelial cells (TEC) in HT, in addition to the known aberrant expression of HLA-DR, coexpress HLA-DQ antigens. Furthermore we provide evidence that class II antigens are synthesized by TEC themselves by demonstration of intracellular HLA-DR gamma-chain. These findings support the theory that TEC may be able to present (auto)antigens in vivo thus perhaps contributing to the perpetuation of thyroid destruction. As expression of class II antigens on TEC was never observed in non- or weakly infiltrated areas, we propose that infiltration by T cells is necessary to induce this aberrant expression of class II antigens.

A variety of immunological, endocrinological, and virological abnormalities have been implicated in the etiopathogenesis of spontaneous autoimmune thyroiditis (SAT) of Obese strain (OS) chickens, e.g., a general T cell hyperreactivity, an increased uptake of iodine into the thyroid gland, a diminution of the glucocorticoid tonus, and an OS-specific endogenous virus. In crosses of the close-bred OS B15/B15 subline with the inbred normal CB B12/B12 strain we have studied the mode of inheritance of these aberrations and their putative association with SAT. The results indicate that none of these OS-specific characteristics alone is an absolute prerequisite for the development of thyroid infiltration, which appears to be governed by one autosomal recessive gene.

Organ-specific autoantibodies (AAb) to thyroid and non-thyroid antigens of various endocrine and exocrine glands (glandular stomach, pancreas, adrenal, parathyroid, and striated muscle) were determined by different serological procedures in sera from Obese strain (OS), Cornell C strain (CS), normal inbred strains (CC and CB), and outbred normal White Leghorn (NWL) chickens. Thyroglobulin autoantibodies (Tg-AAbs), evaluated by immunodiffusion, passive hemagglutination, enzyme-linked immunosorbent assay, and indirect immunofluorescence, as well as other organ-specific AAbs determined by indirect immunofluorescence, predominated in OS chickens. Tg-AAbs were found in the highest frequency, thyroid microsomal AAbs in intermediate frequency, and the other organ-specific AAbs in low frequency in OS chickens. Thyroid and non-thyroid organ-specific AAbs were found only occasionally in control chickens and then only in low titers. Thus, spontaneous autoimmune thyroiditis of OS chickens correlates closely with human Hashimoto thyroiditis not only in respect to AAbs to thyroid antigens but also to nonthyroid organ-specific antigens. Non-organ-specific AAbs, such as antinuclear antibodies, antibodies to chicken red blood cell nuclei, mitochondrial AAbs, smooth muscle antibodies, and reticulin AAbs occur in high frequency in all strains of chickens tested. Even a slight prevalence in NWL chickens was seen, indicating that the abnormal immune response in OS chickens is restricted to organ-specific antigens of the thyroid gland and in some cases also to other exocrine or endocrine glands.

Surgical specimens from patients with Hashimoto thyroiditis (HT) or colloid goiter (CG) were analyzed using an immunofluorescence double staining technique to characterize the infiltrating mononuclear cells (MNC) and to determine the possible expression of HLA-DR antigens by these cells. In HT the majority of infiltrating MNC were T cells. In the interstitium T cells with helper/inducer phenotype (Leu 3a+) were more abundant than those with suppressor/cytotoxic phenotype (OKT8+) and approximately 10-25% of all T cells expressed HLA-DR. Among the cells in peripolesis [i.e., protruding between thyroid epithelial cells (TEC)] OKT8+ cells were observed more frequently than Leu 3a+ cells, expression of DR antigens being 7 and 12%, respectively. The occurrence of Leu 3a+ cells in peripolesis is in marked contrast to the findings in colloid goiter where the intraepithelial population of MNC is almost exclusively composed of OKT8+ cells. The various ways in which the peripoletic Leu 3a+ cells could contribute to the special pathogenesis of HT are discussed.