Search

The most common complication of silicone breast implants is capsular contracture (massive scar formation around the implant). We postulate that capsular contracture is always a sequel to inflammatory processes, with both innate and adaptive immune mechanisms participating. In general, fibroblasts and macrophages have been used as cell types to evaluate in vitro the biocompatibility of breast implant surfaces. Moreover, also T cells have been found at the implant site at the initial stage of fibrous capsule formation. However, only few studies have addressed the influence of surfaces with different textures on T-cell responses. The aim of the present study was to investigate the immune response of human peripheral blood mononuclear cells (PBMC) to commercially available silicone breast implants in vitro. PBMC from healthy female blood donors were cultured on each silicone surface for 4 days. Proliferation and phenotype of cultured cells were assessed by flow cytometry. Cytokine levels were determined by multiplex and real-time assay. We found that silicone surfaces do not induce T-cell proliferation, nor do they extensively alter the proportion of T cell subsets (CD4, CD8, naïve, effector memory). Interestingly, cytokine profiling identified matrix specific differences, especially for IL-6 and TNF-α on certain surface topographies that could lead to increased fibrosis.

Objectives: Systemic sclerosis (SSc) is a chronic connective tissue disease characterized by vascular damage, autoimmunity, and excessive collagen deposition. Despite advances in disease-specific treatment of other rheumatologic diseases, disease-targeted treatment in SSc continues to be elusive. In this review, our goal was to place the contemporary immunobiology of SSc in the perspective of clinical medicine., Methods: We performed a PubMed search for the period from 1989 to 2007, using the keyword, "systemic sclerosis," resulting in a total of 9099 publications, including 1252 reviews. Articles were then selected based on their discussion of recent advances in the elusive pathogenesis of SSc. A final total of 259 articles were chosen for the review., Results: The SSc hallmarks of vascular damage, immunologic activation, and collagen deposition can be traced to 4 major factors: T-cells, fibroblasts, B-cells, and cytokines/chemokines. T-cells are a major component of the infiltrate in skin and lung, exhibiting increased expression of activation markers and showing signs of antigen-driven expansion. Preliminary data indicate that induction of oral tolerance with collagen, a target of SSc T-cell responses, is associated with clinical benefits. Although this suggests that T-cells participate in the pathogenesis of SSc, their precise role and antigen specificity largely remain to be elucidated. Defective numbers and functions of certain T-cell subsets, such as natural killer and gammadelta T-cells, may be involved in the failure to maintain tolerance. Other data suggest that gammadelta T-cells may themselves be effector cells in endothelial cell cytotoxicity. There are several lines of evidence for a pathogenic role of B-cells in SSc, in particular, through the production of autoantibodies. Antibody-dependent cell-mediated cytotoxicity is a primary pathogenic event in an animal model of SSc and is likely to be involved in human SSc. Nonetheless, there is as yet no convincing evidence for the pathogenicity of SSc-specific antibodies. SSc fibroblasts exhibit a specific phenotype characterized not only by excessive collagen production but also by increased responsiveness to and production of cytokines and chemokines. This phenotype is induced by a complex network of cytokines and chemokines but appears to be maintained in the absence of exogenous stimuli via the autocrine production of some of these factors by SSc fibroblasts themselves, particularly transforming growth factor, platelet-derived growth factor, monocyte chemoattractant protein 1, and interleukin-1., Conclusions: Significant variations in laboratory data among patients suggest that the pathology reflects a heterogeneous disease. Nonetheless, the possibility of achieving clinical benefits by inducing oral tolerance highlights the importance of characterizing SSc T-cell antigens. It is hoped that the identification of some of the key players in the induction and maintenance of the SSc fibroblast phenotype may yield new disease-targeted treatment regimens for patients with SSc.

Background: Anti-heat-shock protein 60 (HSP60) antibody-levels have been linked to carotid atherosclerosis and cardiovascular risk in a variety of studies. The potential role of cellular immune reactions against HSP60 has so far attracted little attention in epidemiological research., Methods and Results: In vitro T-cell reactivity to various HSP60s and tuberculin was assessed in blood samples from a elderly subpopulation of the Bruneck study (100 men, 50-69 years) and the young participants of the ARMY study (141 men, 17-18 years), and analyzed for a potential association with common carotoid artery intima-media thickness (IMT). In vivo skin reaction against tuberculin was recorded in subjects of the Bruneck study and correlated with the in vitro proliferative response to tuberculin (P=0.004). T-cells isolated from peripheral blood of all individuals proliferated upon stimulation with HSP60s. In multivariate linear regression analysis adjusted for standard risk factors, T-cell stimulation was significantly related to IMT in the ARMY (P=0.005 for human HSP60 and P=0.064 for mycobacterial HSP60) but not in the Bruneck study., Conclusions: T-cell reactivity against HSP60s correlated with IMT in male youngsters but not in men aged 50 and over, indicating a more prominent role of specific cellular immunity to HSP60s in the young and very early stages of atherosclerosis.

Glucocorticoid hormones are known to act as important modulatory factors in the development of autoimmune diseases, and to play an important role in thymic T-cell selection. There seems to be a finely balanced equilibrium between the apoptosis-inducing effects of glucocorticoid and T cell receptor ligand binding. Here we are investigating whether glucocorticoid-induced T cell apoptosis is mainly dependent on circulating glucocorticoid levels or if the thymus itself is able to produce glucocorticoids. To this end, we attempted to demonstrate enzyme activities of the whole set of steroidogenic enzymes for the synthesis of glucocorticoids in murine thymic tissue. We isolated steroidogenic organelles from thymic tissue, incubated these with radioactive (precursor) steroids in vitro, and visualized the resulting products by thin-layer chromatography. Our results show that the thymus possesses all enzymes and cofactors required for glucocorticoid production. However, an intact thymic architecture is necessary for glucocorticoid production, since 11beta-hydroxylase was not detected in irradiated thymi or in a thymic epithelial cell line. The results of these experiments show that the whole glucocorticoid metabolism takes place within the thymus. This finding provides the biochemical basis for the in situ effects of glucocorticoid hormones on thymocyte development and selection.

Previous studies in our laboratory have shown that arteriosclerotic changes can be induced in normocholesterolemic rabbits by immunization with mycobacterial heat shock protein (hsp) 65. To further investigate the immunologic mechanisms underlying such vascular lesions, 39 male New Zealand White rabbits were treated by triple immunization with fortified Freund's complete adjuvant containing 5 mg/mL Mycobacterium tuberculosis as a source of hsp65 and simultaneous immunosuppressive therapy twice per week with either anti-CD3 monoclonal antibody (1 mg/kg) and prednisolone (1 mg/kg) or prednisolone (1 mg/kg) alone. Sixteen weeks after the first immunization the animals were killed, and as expected, severe arteriosclerotic lesions in the intima of the aortic arch were found in 9 of 10 immunized rabbits. However, only 1 of 10 rabbits immunized and immunosuppressed with the combined anti-CD3 monoclonal antibody and prednisolone treatment showed a single moderate lesion in the aorta, whereas 5 of 9 rabbits immunized and immunosuppressed by prednisolone treatment alone showed lesions, albeit mild. In conclusion, the early inflammatory stages of arteriosclerotic lesions induced by immunization with hsp65 can be inhibited by immunosuppressive therapy with anti-CD3 monoclonal antibody.

Thymocytes form unique multicellular complexes with epithelial cells (thymic nurse cells, TNC) and rosettes (ROS) with macrophages, epithelial cells and dendritic cells. To investigate the role of differentiation checkpoints in the formation of the thymic heterotypic complexes in vivo, we used mutant mice which have genetically defined blocks at early and late stages of T cell development. We show that RAG-1-/-, TCRbeta-/- , and p56lck-/- mice lack thymocyte ROS formation with epithelial cells, macrophages, or dendritic cells. TNC formation was not affected by TCRbeta and p56lck gene mutations but partially decreased in RAG-1-/- mice, indicating that TNC are the earliest thymocyte-stromal cell complexes formed in development, whereas ROS only appear after thymocytes have rearranged and expressed a functional TCRbeta chain. Genetic blocks in CD8 lineage commitment (CD8-/- and IFN regulatory factor-1-/- mice) and positive and negative T cell selection (CD45-/-, TCRalpha-/-, and CD30-/- mice) did not affect thymocyte-stromal cell complexes. Surprisingly, CD4-/- mice, but not MHC class II-/- mice, had significantly reduced numbers of TNC and ROS, in particular, a severe defect in ROS formation with thymic dendritic cells. The CD4-/- block in ROS and TNC formation was rescued by the introduction of a human CD4 transgene. Moreover, we show that the adhesion receptors CD44 and LFA-1 cooperate in the formation of the thymic microenvironment. These results provide genetic evidence on the role of defined stages in T cell development and adhesion molecules on thymocyte/stromal cell interactions in vitro.

Thymic nurse cells (TNC) and T-cell stromal rosettes (ROS) are two in vivo models for stromal cell-thymocyte interactions. We describe a simplified enrichment method for TNC and ROS that overcomes the necessity for large amounts of tissue. The complexes were further analyzed with confocal microscopy, and three subunits of ROS were defined on the basis of their central cell phenotype, i.e., macrophage, dendritic, or epithelial cell rosettes. Because adhesion molecules are proposed to play a crucial role in T-cell development, we investigated CD44, LFA-1, and ICAM-1 expression in such complexes. The epithelial component of TNC expresses CD44 and ICAM-1, whereas intra-TNC thymocytes are LFA-negative. With regard to ROS, all subsets expressed CD44, and macrophage and dendritic cell ROS were also ICAM-1-positive and LFA-1-positive. The current protocol opens the possibility for further in vivo analysis of stromal cell-thymocyte interactions, e.g., for studies of scarce gene mutant mice.

Bacterial cell-wall lipopolysaccharide (LPS) is the main endotoxin contributing to local inflammation and systemic toxicity during Gram-negative infections and induces aortic endothelial injury with or without cell death and replication followed by increased leukocyte adhesion. Heat-shock protein (hsp) 60 is under study in our laboratory as a potential antigen inducing immunologic attack to endothelial cells in atherogenesis. To investigate the mechanism of LPS-induced endothelial injury and the phenotypes of adhering cells, Lewis rats were treated in vivo or, in aortic organ cultures, with LPS to determine the expression of intercellular-adhesion molecule-1 (ICAM-1) and hsp60 on aortic endothelium and to characterize phenotypes of adhering leukocytes. Increased ICAM-1 expression by aortic endothelium was observed as early as 3 hr after LPS injection and persisted up to 72 hr, whereas elevated levels of hsp60 were found between 6 and 48 hr. In vitro application of various types of stress, such as LPS, H2O2, and high temperature, not only stimulated endothelial expression of hsp60 but, concomitantly, that of ICAM-1. The number of adhering leukocytes was significantly increased on aortic endothelium 6 hr after LPS administration, and the predominant leukocytes adhering to stressed endothelium were monocytes (80%) and T lymphocytes (8 to 20%). In organ cultures of rat aortic intimal, LPS, and H2O2 evoked increased leukocyte adhesion, which proved to be selective, because adherent leukocytes were mostly Ia+ monocytes and T cells, i.e., activated. Adhering T cells were gamma/delta antigen-receptor positive in 8 to 16% after LPS stress, whereas these cells amount to only 2 to 4% of peripheral blood T cells. Blocking of adhesion molecules ICAM-1, LFA-1 alpha, and/or LFA-1 beta reduced adhesion up to 34%. Increased coordinated LPS-dependent expression of hsp60 and ICAM-1 correlates with monocyte and T-cell adhesion to aortic endothelium. These observations may be significant for elucidating the mechanism of the initiating events in the development of atherosclerosis.

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.

Graves' ophthalmopathy is an autoimmune condition characterized by T cell infiltration of the retrobulbar tissue. Phenotypic and functional analysis of these infiltrating cells may provide insight into the pathogenesis of the disease. IL-2-responsive cells were therefore grown out of the retrobulbar tissue from two patients with severe Graves' ophthalmopathy undergoing orbital decompression surgery, and six T cell lines were established and characterized. They consisted predominantly of CD8 + CD45RO+ cells and secreted IL-4, IFN-gamma, and IL-10 upon activation. When screened for their antigen reactivity, all lines proliferated in response to stimulation with autologous retrobulbar fibroblasts in an HLA class I-restricted manner, but did not recognize autologous peripheral blood mononuclear cells, crude eye muscle extract, allogeneic cells, or purified protein derivate of Mycobacterium tuberculosis. In contrast, PBMC from the same patients responded readily to purified protein derivate of Mycobacterium tuberculosis and allogeneic PBMC, but did not recognize autologous fibroblasts. Interestingly, only one of the six retrobulbar T cell lines displayed cytotoxicity towards its specific target cell population. These results suggest that the retrobulbar fibroblasts are a major T cell target in Graves' ophthalmopathy. Pronounced cytokine production in the absence of target cell cytotoxicity may explain fibroblast proliferation, glycosaminoglycan secretion, and secondary eye muscle enlargement in this condition.

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.

We have shown previously that atherosclerotic lesions can be induced in normocholesterolemic rabbits by immunization with mycobacterial heat shock protein 65 (hsp65), which has a high degree of sequence homology with mammalian hsp60. To investigate a possible relationship between hsp60 expression and the antigenic specificities of infiltrating T cells in the lesion, 38 New Zealand White rabbits were treated either by immunization with recombinant mycobacterial hsp65 or by administration of a 0.2% cholesterol diet. Atherosclerotic lesions were observed after 16 wk, particularly in the aortic arch and arterial bifurcations of rabbits immunized with hsp65 or fed with a cholesterol-rich diet. Hsp65 staining of aortas showed a heterogeneous distribution, and significantly increased staining intensity in atherosclerotic lesions compared to aortic media or adventitia. This abundantly expressed hsp65 was observed in atherosclerotic lesions induced by hsp65 immunization as well as those induced by cholesterol-rich diet alone. Interestingly, a population of the T lymphocytes isolated from all forms of atherosclerotic lesions specifically responded to hsp65 in vitro. IL-2-expanded T cell lines derived from atherosclerotic lesions showed a significantly higher hsp65 reactivity than those developed from peripheral blood of the same donor. Furthermore, levels of circulating antibodies and numbers of spleen cells specifically reacting against hsp65 were elevated in all experimental animals. Flow cytometric analysis of spleen cells showed elevated immune response-associated antigen expression in treated animals. In conclusion, increased hsp65 expression in intimal cells and the presence of hsp65-specific T cells in blood and in atherosclerotic lesions may be important in initiating the development of atherosclerosis and perpetuating the lesions.

It has been postulated that thymic nurse cells (TNC), lymphoid-epithelial complexes composed of thymocytes enclosed within major histocompatibility complex (MHC) class I+ and class II+ cortical epithelial cells, may provide an optimal microenvironment for the process of T cell selection. By transplanting single TNC in the avian chorionallantoic membrane assay we demonstrate that a significant portion of intra-TNC lymphocytes (TNC-L) possess reactivity against self-MHC molecules. The frequency of these autoreactive cells among TNC-L exceeds by far that of thymocytes or peripheral blood lymphocytes of the same donor. These results indicate that TNC-L constitute a T cell population enriched for self-MHC reactivity, i.e. cells that have undergone positive selection, but not yet deletion and/or deactivation.

The role of plasma lipoproteins in atherogenesis is well recognized but the physiological relevance of their immunoregulatory properties is still questioned. Here Karine Traill and colleagues outline the recent advances that have been made towards unravelling the mechanisms of immunoregulation by lipoproteins in vitro and consider whether any of these mechanisms are operative in vivo. In particular they address the possible detrimental effects of high serum lipoprotein levels on immune function and the question of whether hyperlipidemia (or hypercholesterolemia) should be considered a risk factor for diminished immunity, for example in old age.

Single chicken thymic nurse cells (TNC) placed onto the chorionallantoic membrane (CAM), showed that intra-TNC lymphocytes (TNC-L) possess a strong graft-versus-host reactivity (GVHR) in allogeneic MHC combinations. This reaction shows the morphological, phenotypic, and functional characteristics of a classical GVH reaction (GVHR). The induction of a GVHR was significantly higher for TNC-L as compared with thymocytes or peripheral blood lymphocytes (PBL). The specificity of the GVHR was shown by serial transfer experiments onto appropriate allogeneic and syngeneic secondary embryonic hosts. In immunofluorescence analyses with monoclonal antibodies (mAb) to the chicken alpha/beta and gamma/delta T cell receptors (TCR) and the CD3, CD4, and CD8 equivalents, an enrichment of CD3+/CD4+/CD8- and CD3+/CD-4-/CD8+, TCR-alpha/beta + and TCR- gamma/delta + cells was observed inside TNC as compared with extra-TNC thymocytes. A large proportion of CD4+ and/or CD8+ TCR- gamma/delta + cells were demonstrated inside TNC. A minor population among TCR- gamma/delta extra-TNC thymocytes also expressed CD4 and/or CD8 molecules. Based on functional tests and double staining experiments, we propose that CD4+/CD8+ thymocytes enter the TNC where they may undergo positive selection for MHC restriction and further differentiation to CD4 or CD8 single-positive cells. Taken together these data support the concept that TNC contribute a specialized thymic microenvironment for T cell differentiation and maturation.

Like all cells, lymphocytes need cholesterol for proper function, a requirement met by a finely tuned homeostasis between intracellular synthesis and uptake from the environment via low-density lipoproteins (LDL). We used flow cytometry to analyze the receptor activity of resting cells and T blasts incubated/activated in serum-free culture medium, or in medium supplemented with 25-5,000 micrograms/ml LDL. Dioctadecyl-indocarbocyanine has proved to be a useful fluorescent probe for investigating the LDL receptor activity of lymphocytes. The results show the receptor activity of day-3 resting T cells to be reduced more than 50% by 50 microgram LDL/ml, whereas 100-fold higher concentrations are necessary to achieve the same level of reduction in day-3 PHA blasts. The LDL receptor activities of individual blood donors' resting T cells, in vitro cholesterol-deprived resting T cells, and activated T blasts, were compared using two analytical techniques: spectrofluorometric analysis of detergent-solubilized cell suspensions and flow cytometric analysis of single living cells. Receptor affinity was determined by Scatchard analysis of spectrofluorometric binding curves, and by Line-weaver-Burke plots of flow cytometric data. Both methods yielded essentially identical dissociation constants (Kd) for cholesterol-deprived resting T cells and mitogen-activated T blasts, which fell in the expected range for the high-affinity LDL receptor (4.1-8.9 nM). In addition, spectrofluorometric analysis, but not flow cytometry, permitted quantification of LDL uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

A high density lipoprotein (HDL) receptor/binding site has been identified on peripheral blood lymphocytes, and some of its properties were compared with those of HDL receptors on other cell types. Binding studies were performed using fluorescent (dioctadecylindocarbocyanine)-labelled HDL (DiI-HDL) and analyzed by fluorescence microscopy and quantitative flow cytometry. Uptake of low levels of DiI-HDL during a 2 h incubation at 37 degrees C was a property of all lymphocytes, i.e. not of one particular subset only. Visual inspection of these cells in the fluorescence microscope revealed both membrane and cytoplasmic fluorescence, indicating that DiI-HDL become internalized during the 2 h incubation; internalization appeared to be a receptor-mediated process. In competitive binding studies, apo E-free HDL competed effectively for DiI-HDL binding, whereas LDL competed very weakly. Two features of DiI-HDL uptake are demonstrated which are unique to lymphocytes: 1) it was enhanced 3-6-fold by inclusion of EDTA in the incubation medium or by incubating in Ca2+/Mg2+ free medium, and 2) it was saturable at 37 degrees C.

Thymic nurse cells (TNC) are multicellular complexes composed of morphologically intact thymocytes internalized within epithelial cells. Due to their unusual features, they have been suspected to provide an appropriate microenvironment for T-cell repertoire acquisition. In recent functional studies in the chicken system, we have shown that intra-thymic nurse cell-lymphocytes (TNC-L) are highly efficient inducers of a graft-versus-host reaction (GvHR) in an allogeneic chorionallantoic membrane (CAM) assay, manifested by the formation of visible colonies, so-called pocks. Immunohistochemical and electron microscopical assessment of pocks revealed no differences between the cellular components and ultrastructural features of GvHR induced by TNC-L or peripheral blood lymphocytes (PBL). In addition, the stimulation of this local GvHR by interleukin 2 (IL-2) was demonstrated.

Chicken thymic nurse cells (TNC) were isolated and their plasma membrane antigens were characterized by immunofluorescence with a panel of allo-, hetero-, and monoclonal antibodies. Analogous to the murine system, chicken TNC are localized in the subcapsular region of the cortex and were strongly B-L and B-F positive (MHC Ia and H-2 D, K equivalents). The TNC were non-phagocytic and lacked fc receptors, but expressed an antigen specific for thymic epithelial cells, and their cytoplasm was stained by an anti-keratin antiserum. The internalized lymphocytes were all intact T cells of a distinct differentiation stage, their antigenic profile consistent with that of immature cortical thymocytes. To determine whether TNC may play a role in the activation of autoimmune disease, Obese strain chickens with spontaneous autoimmune thyroiditis were investigated. These chickens were found to have a twofold defect in their TNC content: the thymuses contained approximately one-half of the number of TNC found in normal chickens beginning as early as 17 days of embryogenesis, and the lymphocyte content of the Obese strain TNC was one-half that of the normals. The deficiency is specific for TNC and may be a major reason for the abnormal T cell differentiation in the Obese strain.

The aim of this study was the delineation of different antigenic surface determinants on the surface of adult peripheral T cells by means of a specific horse anti-human T-cell serum (ATS). It was shown that this serum reacts both with E receptors and (an) additional T antigen(s). While E receptors showed the already known susceptibility to trypsin, T antigens (as demonstrated in cytotoxicity tests) were resistant to trypsinization even at high concentration. Incubation of the trypsinized peripheral blood lymphocytes (PBL) in 5 per cent CO2 allowed the resynthesis of E-receptors. High concentrations of ATS (without complement) significantly inhibited rosette-formation. This suggests a close steric relationship between E receptors and T antigen(s). However, absorptions of ATS with trypsinized PBL left the E-rosette inhibitory capacity unaltered. Treatment of PBL with ATS in appropriate dilutions and indirect immunofluorescence tests under capping conditions followed by conventional rosette procedures showed that the E receptor and T antigens are separately mobile within the T-cell membrane.

Spontaneous autoimmune thyroiditis of OS chickens is associated with a marked hyperreactivity of the T cell system. The purpose of the present study was to investigate the underlying regulatory mechanisms. Co-cultivation experiments between Con A-stimulated OS and NWL lymphocytes in communicating cultures revealed soluble regulatory factors to be responsible for the observed functional differences: the high proliferative response to Con A and hyperproduction of IL 2 of OS cells was found to be due to a deficiency in the conditioned medium of dialyzable inhibitory factor(s) that regulate IL 2 secretion of NWL lymphocytes. Furthermore, sera of young NWL chickens were found to profoundly inhibit the IL 2-promoted lymphoblast proliferation. This IL 2 antagonizing activity is lost with age (3 to 6 yr) and was found to be significantly diminished in OS birds throughout ontogeny, thus pointing to possible parallels between immune regulatory dysfunction in autoimmunity and in physiologic aging. Both enhanced T cell response and the defect in serum suppressor were inherited by (OS X CB)F1 animals, indicating that these two aberrations may be related to each other.

This paper is intended to demonstrate the extension of experimental procedures used for the production of anti-chicken T-cell sera to the manufacturing of anti-human T-cell sera. Specific anti-chicken bursa (ABS) and anti-chicken thymus cell sera (ATS) were prepared in turkeys. The in vitro specificity of these sera was assessed by means of immunofluorescence and lymphocytotoxicity tests. The selective immunosuppressive effect of ABS was demonstrated in Obese strain (OS) chickens with spontaneous autoimmune thyroiditis, that of ATS in a skin allograft system. A specific anti-human T-cell serum was prepared by immunizing a horse with fetal human thymus cells. After appropriate exhaustive absorptions this serum was found to react specifically with human T-cells. An anti-T cell globulin (ATG) fraction prepared from the absorbed antiserum was then labelled with FITC for use in direct immunofluorescence tests. The potential diagnostic value of T-cell specific antibodies is discussed.

The enhanced T cell reactivity (ConA hyperresponsiveness and IL 2 hypersecretion) of spleen lymphocytes of Obese strain (OS) chickens with spontaneous autoimmune thyroiditis has recently been shown to be due to a defect in macrophage-derived non-specific suppressor factors that regulate IL 2 secretion and IL 2-promoted T lymphoblast proliferation in normal healthy animals. In the present study, we present several lines of evidence that the increased T cell response of peripheral blood lymphocytes (PBL) of OS chickens is due to mechanisms entirely different from the described dysregulation of splenic T cells: 1) In contrast to the splenic macrophages, peripheral blood monocytes of OS animals are not deficient in the production of IL 2 antagonistic activity (IAA); 2) therefore, cocultivation of PBL from OS and Normal White Leghorn (NWL) chickens in communicating culture chambers did not abrogate the difference in Con A response as previously observed with spleen lymphocytes. 3) Immunofluorescence with a monoclonal antibody (INN CH 16) against the chicken IL 2 receptor revealed enhanced numbers of mitogen activatable T cells in OS PBL but not OS spleen lymphocytes. 4) After prolonged Con A stimulation of PBL, OS and NWL lymphoblasts did not differ from each other in functional aspects. In contrast to this, Con A lymphoblasts from OS spleens exhibited enhanced staining with INN CH 16 in parallel with an increased proliferative response to IL 2. Thus, the primary T cell dysfunction involved in the development of autoimmune disease in OS chickens is the result of at least two separate regulatory defects.

The possibilities for functional analysis of thymic nurse cells (TNC) have so far been hampered by the low yield of these cells by means of gradient centrifugation and their fragility preventing purification by a fluorescence-activated cell sorter. Furthermore, they are extremely unstable in cell culture. Here we present functional data obtained by the analysis of single TNC in an avian system: the chicken with its extramaternal embryonic development prompted us to use the chorioallantoic membrane (CAM) pock formation assay to investigate the possible graft-vs.-host reactivity (GVHR) of TNC lymphocytes (TNC-L). The CAM assay proved to be an optimal and unique "cell culture" system for the investigation of the functional characteristics of TNC. Our data show that TNC-L, unexpectedly, are able to exert a GVHR on allogeneic CAM with the very high efficiency of 1/13 as compared to thymocytes of the same donors showing a GVHR efficiency of 1/350. TNC-L are even able to induce GVH-like reactions in syngeneic combinations, while thymocytes and peripheral blood lymphocytes from the same donors always remain negative.

The mitogenic response to Con A and the production of T cell growth factor or interleukin 2 (IL 2) by splenic and peripheral blood lymphocytes of obese strain (OS) chickens with spontaneous autoimmune thyroiditis have been investigated. By using an optimized method with Con A-coated chicken erythrocytes (MRC), lymphocytes of OS chickens were found to exhibit significantly elevated mitogenic responses as compared with cells from either Normal White Leghorn chickens (NWL) or animals of the Cornell C-Strain (CS), from which the OS has originally been developed. This difference was observed throughout ontogeny up to 15 mo of age, and was associated with increased levels of IL 2 activity in the culture supernatants. The elevated responsiveness of OS T lymphocytes was also found to be manifested in the expression of receptors for IL 2, because Con A-stimulated lymphocytes of OS birds were significantly more effective than those from normal controls in absorbing IL 2 activity from conditioned media (CM) of stimulated spleen cells. High concentrations of CM were suppressive in IL 2 assays, signaling the presence of an inhibitory factor(s) in addition to IL 2. An additional indication for defective immunoregulation was that CM from OS lymphocyte cultures showed significantly less of this suppressive activity in comparison with CM of normal (NWL and CS) lymphocyte cultures. Finally, the spontaneous uptake of 125IUdR of embryonic and early post hatching OS spleen lymphocytes was consistently and significantly enhanced. This difference, however, in contrast to the one observed in Con A responses, was found to decrease with age. The data are discussed in view of the contradictory results concerning T cell functions reported for several autoimmune states in mammals.

A combination of neonatal thymectomy and intensive treatment with a highly specific turkey anti-chicken T cell serum effectively abrogates the spontaneous development of thyroiditis and thyroglobulin autoantibodies in the Obese strain chicken. Thus, in addition to a restraining influence of suppressor T cells on the disease process indicated by earlier studies, it would appear that T cells also make a positive contribution to pathogenesis.

ATS contains antibodies of different specificity directed against E receptors and T specific antigens, respectively. E-receptors are trypsin-sensitive, T-antigens are trypsin-resistant. Absorption of ATS with trypsinized thymocytes thus removes anti-T, but leaves anti-E antibodies unaffected. The rosette inhibitory potential of the absorbed antiserum (anti-E) remains unaltered. Immunization with trypsinized thymocytes on the other hand results in the production of anti T-cell sera highly specific in immunofluorescence and cytotoxicity tests without contaminating anti-E antibodies and, therefore, also lacking any rosette inhibitory capacity. E receptors and T antigens are independently mobile within the cell membrane as shown by differential capping.

Lymphocytes were isolated from the infiltrated thyroid glands of 2- to 5-wk-old Obese strain (OS) chickens with spontaneous autoimmune thyroiditis (SAT). Immunofluorescence analysis performed by using a panel of monoclonal and polyclonal antibodies revealed that 60% of thyroid infiltrating leukocytes (TIL) were mature T cells, a large portion of which seemed to be in an activated state bearing Ia-like antigens (10%) as well as a surface determinant associated with T cell activation (16%), i.e., possibly the receptor for interleukin 2 (IL 2). Furthermore, a relatively high plasma cell content (5%) was observed. TIL exhibited high proliferative responses to T cell mitogens (concanavalin A, phytohemagglutinin) and IL 2, but only weak responses to the B cell mitogen LPS from Salmonella typhimurium. When injected into newly hatched, MHC-identical, irradiated normal chickens, TIL induced both the production of autoantibodies and thyroid infiltration. Peripheral lymphocytes from spleen and blood and thymocytes from the same OS donors had no effect. Analysis of chemically (cyclophosphamide) bursectomized OS chickens suggested that an intact B cell system was not obligatory for the induction of SAT. TIL from these chickens consisted of 77% T cells and less than 1% B lymphocytes, yet were capable of inducing severe thyroid infiltration upon transfer into normal recipients. These findings emphasize the importance of the T cell system in the initiation of SAT.

A monoclonal antibody (INN-CH-16) was prepared which reacts with a cell surface antigen termed chicken activated T lymphocyte antigen. This antigen is expressed on antigen- or mitogen-activated T lymphocytes and is not present on nonstimulated lymphocytes. It has an apparent molecular mass of 48-50 kDa under reducing conditions. The value of this antibody for the immunohistochemical characterization of infiltrating cells in the thyroid glands from Obese strain chickens with spontaneous thyroiditis is demonstrated.

In contrast to systemic autoimmunity, spontaneous autoimmune thyroiditis of Obese strain (OS) chickens is associated with a marked T cell hyperreactivity in vitro, i.e. an increased proliferation and interleukin 2 (IL 2) secretion in response to Concanavalin A (ConA). In the present study we report an enhanced capacity of OS peripheral lymphoid cells (splenocytes and peripheral blood lymphocytes, PBL) to adsorb fluorescein isothiocyante (FITC) labelled ConA, but not phytohemagglutinin (PHA). However, the elevated ConA binding cannot be a prerequisite for in vitro ConA hyperreactivity as OS thymocytes are normal with respect to ConA binding but nonetheless exhibit elevated responses to this mitogen. Moreover, ConA binding does not correlate with the frequency of cells able to express IL 2 receptors upon short term ConA stimulation. The percentage of ConA activatable cells was found to be increased in OS- PBL as compared to normal control PBL, but was unaltered in OS splenocytes. This finding points to a further mechanism of T cell hyperreactivity in OS chicks in addition to the previously reported defects in nonspecific immunosuppression. Finally, enumeration of cells in the S phase revealed that enhanced proliferation of OS T lymphocytes was not restricted to the in vitro response to ConA and phytohemagglutinin (PHA) but also occurs in vivo.

Step-wise dilution of chicken thymus cell suspensions has been used to sequentially reveal suppressor, effector, and helper cells in these suspensions. The cells were tested either alone or in autologous mixture combinations with peripheral blood lymphocytes (PBL) as a source of effector cells. The assays studied were graft-vs-host reaction (GvHR) and mixed lymphocyte (MLR) reaction, spontaneous cellular cytotoxicity and antibody-dependent cell-mediated cytotoxicity, and mitogen responsiveness to Con A, PHA, and PWM. When tested alone, high numbers of thymus cells (1 X 10(7) gave weak or low responses, with the exception of GvHR, which was high. When this number of thymocytes was mixed with a strongly responding PBL effector population, there was marked suppression of the latter. Nonspecific crowding was excluded as a cause for the decreased responsiveness, and the data therefore demonstrated the presence of suppressor cells in the thymus. With gradual reduction of the thymus cell number in the mixtures, the suppressor activity was lost, but concomitant with this was the appearance of, or a gradual increase in, thymus effector cells giving good responses. Further dilutions of the thymus (to, e.g., 1 X 10(5) cells) depleted the suspension of effector cells, but helper cells capable of markedly amplifying the effector potential of PBL were revealed. The suppressor/helper function of the thymus was not only dependent on the absolute numbers of thymus cells present, but also on the degree of inherent responsiveness of the effector PBL. If the response of PBL alone was strong, a thymus suspension containing both helper and suppressor cells (e.g., 1 X 10(6) cells) caused suppression of the PBL; if the PBL alone were weak, this same thymus cell suspension caused enhancement. The outcome of an immune response is therefore dependent not only on the presence or absence of particular cell types, but also on the ratios between these cells. An imbalance in these ratios in vivo may underlie diseases of immunologic origin, e.g., autoimmunity.

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.

A correlation has been sought between the effects of lipids on membrane fluidity and mitogen responsiveness of human peripheral blood lymphocytes (PBL). Cholesterol and the reputedly potent membrane fluidizing agent AL721 (1) were used for these studies. However, the large AL721 induced increase in membrane "fluidity", assessed by steady state polarization of the probe 1,6-diphenyl-1,3,5-hexatriene (DPH), was found to be an artefact arising from lipid uptake by monocytes. Mitogen responses were enhanced by AL721 but unaffected by cholesterol. It is concluded that AL721 does not exert its effect through enhanced triggering of cells by altered membrane fluidity but rather that lymphocytes require an exogenous source of phospholipids/triglycerides for optimal growth in vitro, although they can synthesize sufficient cholesterol to meet their own needs.

Appropriately absorbed turkey antisera to antigenic surface determinants of chicken bursa (ABS) or thymus cells (ATS) were assessed for their selective immunosuppressive activy in vitro and in vivo. The intraperitoneal injection of ABS or ATS into 2-3-week-old normal white Leghorn chickens entailed a significant depletion of B or T cells respectively from spleen and peripheral blood, while bursa and thymus themselves remained unaffected. The potential of this 'peripheral serological bursectomy and thymectomy' paralleled that found after the conventional surgical procedures with subsequent sublethal irradiation. The mean survival time of skin allografts from donors of genotype B4B4 onto B8B8 recipients was significantly prolonged by treatment with ATS (29 plus or minus 12 days) as compared to untreated (14 plus or minus 2 days), normal turkey serum (12 plus or minus 3 days) or ABS-injected (13 plus or minus 2 days) groups. This selective suppression of a T cell-dependent immune reaction by ATS was also confirmed in vitro by its inhibitory action on the graft-versus-host reactivity of adult peripheral blood lymphocytes in the chorioallantoic membrane assay, where normal turkey serum and ABS were again ineffective. Thus, ABS or ATS produced in avian species may serve not only to delineate B and T cells in vitro, but can also be used for selective manipulation of immune reactions of the chicken in vivo.

The disturbed homeostasis of the immune system in Obese strain (OS) chickens with spontaneous autoimmune thyroiditis consists in a general T cell hyperreactivity (concanavalin A hyperresponsiveness, interleukin 2 (IL 2) hypersecretion), particularly expressed by those lymphocytes which infiltrate the diseased thyroid gland. This abnormality has been attributed to a defective regulation of both IL 2 production and IL 2 function by low m.w. factors, which are present in serum and splenocyte culture supernatants of normal chickens, but deficient in the OS. In the present study we identified the cellular origin of IL 2 antagonistic activity as a nonlymphoid, adherent cell. Suppressor factor production in vitro was confined to the plastic adherent fraction of spleen cells and preincubation of splenocytes with nylon wool, silica particles, or carbonyl iron significantly reduced the nonspecific suppressive activity of the culture supernatant. Kinetic studies revealed the defect in nonspecific suppression to entail prolonged IL 2 production by concanavalin A-activated OS spleen cells. In vivo treatment of normal White Leghorn chickens with silica led to a decrease in suppressive serum activity down to the OS level, whereas neither neonatal thymectomy nor bursectomy had any effect. The defective suppressor factor production in autoimmune chickens appeared to be due to a functional, but not numeric defect of macrophages as revealed by phenol red staining. The possibility that this aberration in adherent cell function might be a secondary phenomenon to the recently described reduced corticosterone tonus in OS chickens was excluded by in vivo substitution with exogenous glucocorticoids which did not normalize the suppressor defect in serum or in conditioned medium. Finally, we present evidence that T lymphoblasts from OS animals are less susceptible to IL 2 antagonistic regulation than normal cells, which possibly further contributes to the T cell hyperfunction of this autoimmune strain.

Three subsets of chicken peripheral T cells (T1, T2 and T3) have been identified in peripheral blood of adult chickens on the basis of fluorescence intensity after staining with certain xenogeneic anti-thymus cell sera (from turkeys and rabbits). They differentiate between 3-10 weeks of age in parallel with development of responsiveness to the mitogens concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM). Functional tests on the T subsets, sorted with a fluorescence-activated cell sorter, have shown that T2, 3 cells respond to Con A, PHA and PWM and are capable of eliciting a graft-vs.-host reaction (GvHR). In contrast, although T1 cells respond to Con A, they respond poorly to PHA and not at all to PWM or in GvHR. There was some indication of cooperation between T1 and T2,3 cells for the PHA response. Parallels between these chicken subsets and helper and suppressor/cytotoxic subsets in mammalian systems are discussed.