Onelia Bistoni

CD4+CD28− T Lymphocytes Contribute to Early Atherosclerotic Damage in Rheumatoid Arthritis Patients

Background—Peripheral blood expansion of an unusual CD4+ T-cell subset lacking surface CD28 has been suggested to predispose rheumatoid arthritis (RA) patients to develop more aggressive disease. However, the potential association between CD4+CD28null T cells and early atherosclerotic changes in RA has never been investigated. Methods and Results—The number of circulating CD4+CD28null cells was evaluated in 87 RA and 33 control subjects who also underwent evaluation of carotid artery intima-media thickness (IMT) and endothelial function via flow-mediated vasodilation (FMV). Patients had higher IMT and lower FMV compared with control subjects. The frequency of CD4+CD28null cells was significantly higher in patients than in control subjects. Twenty patients with persistent expansion of circulating CD4+CD28null cells had more marked increase of carotid artery IMT and stronger decrease of brachial artery FMV. Blockade of tumor necrosis factor-&agr; led to a partial reappearance of the CD28 molecule on the CD4+ cell surface. Conclusions—Circulating CD4+CD28null lymphocytes are increased in RA. Patients with persistent CD4+CD28null cell expansion show preclinical atherosclerotic changes, including arterial endothelial dysfunction and carotid artery wall thickening, more significantly than patients without expansion. These findings suggest a contribution of this cell subset in atheroma development in RA. Moreover, the demonstration that tumor necrosis factor-&agr; blockade is able to reverse, at least in part, the CD28 deficiency on the CD4+ cell surface may be of interest for possible innovative therapeutic strategies in cardiovascular diseases.

Human CD1-restricted T cell recognition of lipids from pollens

Plant pollens are an important source of environmental antigens that stimulate allergic responses. In addition to acting as vehicles for foreign protein antigens, they contain lipids that incorporate saturated and unsaturated fatty acids, which are necessary in the reproduction of higher plants. The CD1 family of nonpolymorphic major histocompatibility complex–related molecules is highly conserved in mammals, and has been shown to present microbial and self lipids to T cells. Here, we provide evidence that pollen lipids may be recognized as antigens by human T cells through a CD1-dependent pathway. Among phospholipids extracted from cypress grains, phosphatidyl-choline and phosphatidyl-ethanolamine were able to stimulate the proliferation of T cells from cypress-sensitive subjects. Recognition of phospholipids involved multiple cell types, mostly CD4+ T cell receptor for antigen (TCR)αβ+, some CD4−CD8− TCRγδ+, but rarely Vα24i + natural killer–T cells, and required CD1a+ and CD1d+ antigen presenting cell. The responding T cells secreted both interleukin (IL)-4 and interferon-γ, in some cases IL-10 and transforming growth factor-β, and could provide help for immunoglobulin E (IgE) production. Responses to pollen phospholipids were maximally evident in blood samples obtained from allergic subjects during pollinating season, uniformly absent in Mycobacterium tuberculosis–exposed health care workers, but occasionally seen in nonallergic subjects. Finally, allergic, but not normal subjects, displayed circulating specific IgE and cutaneous weal and flare reactions to phospholipids.

Chemokines, sTNF-Rs and sCD30 serum levels in healthy aged people and centenarians.

Several lines of evidence point to a profound remodelling of the cytokine network in healthy elderly subjects, with decreased type-1 cytokine production (IL 2) and a shift to type 0 and 2. We have also observed an increase of proinflammatory cytokines (IL-1, IL-6, TNF-alpha) in vitro, and an increase of circulating stem cell factor in vivo. In this setting, we studied changes of chemokines (MCP-1 and RANTES) with aging, as well as other molecules, namely, sTNF-RI and sTNF-RII, and the soluble form of the CD30 molecule (sCD30), involved in the pro- and antiinflammatory cytokine balance. The subjects enrolled in the study belonged to three different selected healthy groups of young, aged and centenarians. The presence of rheumatoid factor (RF) and antinuclear antibodies (ANA) was simultaneously assessed. The results show that MCP-1 serum levels were higher in the healthy aged and lowest in the young, while RANTES increased exclusively in centenarians. Only centenarians had autoantibodies (ANA and RF). sTNF-RI and sTNF-RII were significantly elevated in healthy old subjects compared to the young, and even higher in selected centenarians compared to the other age groups. sCD30 serum levels were significantly raised in centenarians compared to the young, despite absence of circulating CD30+ cells in the peripheral blood of the whole study population. No relationship among serum values of these different members of the TNF-R family was found, despite a strong correlation for sTNF-RI and sTNF-RII in all groups. We hypothesize that the increased chemokine levels in aged people, and raised sCD30 levels in centenarians, may reflect a general shift towards type 0/2 cytokines in normal aging, which may be responsible, at least in part, for the appearance of circulating autoantibodies without definite clinical consequences at advanced age.

Balance between Regulatory T and Th17 Cells in Systemic Lupus Erythematosus: The Old and the New

Pathogenic mechanisms underlying the development of systemic lupus erythematosus (SLE) are very complex and not yet entirely clarified. However, the pivotal role of T lymphocytes in the induction and perpetuation of aberrant immune response is well established. Among T cells, IL-17 producing T helper (Th17) cells and regulatory T (Treg) cells represent an intriguing issue to be addressed in SLE pathogenesis, since an imbalance between the two subsets has been observed in the course of the disease. Treg cells appear to be impaired and therefore unable to counteract autoreactive T lymphocytes. Conversely, Th17 cells accumulate in target organs contributing to local IL-17 production and eventually tissue damage. In this setting, targeting Treg/Th17 balance for therapeutic purposes may represent an intriguing and useful tool for SLE treatment in the next future. In this paper, the current knowledge about Treg and Th17 cells interplay in SLE will be discussed.

High levels of the soluble form of CD30 molecule in rheumatoid arthritis (RA) are expression of CD30+ T cell involvement in the inflamed joints

The CD30 is a surface molecule expressed by Th2‐type lymphokine‐producitig T cells upon activation. CD30‐expressing activated T cells release a soluble form of the molecule, which can be detectable both in vitro and in vivo. In the present study, high levels of soluble CD30 were found in peripheral blood and synovial fluid from patients with RA. However, CD30+ CD3+ cells, either CD4+ or CD8+, were significantly present in synovial fluid, but not in peripheral blood, of RA patients. Serum values of soluble CD30 were higher in active than inactive RA patients and directly correlated with rheumatoid factor serum titres. These data strongly support an involvement of CD30+ T cells in the immune processes of rheumatoid synovitis, and may suggest a relationship between Th2‐type cytokine‐secreting T cells and the pathological response in RA.

Role of CD30+ T cells in rheumatoid arthritis: a counter-regulatory paradigm for Th1-driven diseases

CD30 has been proposed to identify Th0/2-type clones. However, the in vivo relevance of this finding is still a matter of debate, as high serum levels of soluble CD30 have been found in both Th1- and Th2- dominated disorders. Among these, rheumatoid arthritis represents a condition where the Th1 predominance is combined with the presence of CD30(+) T-cell activity, particularly in specific stages of the disease. This article discusses the hypothesis that CD30(+) T cells might play a counter-regulatory role at sites of inflammation in Th1-mediated conditions, such as rheumatoid arthritis.

Increased Allergen-Specific, Steroid-Sensitive γδ T Cells in Bronchoalveolar Lavage Fluid from Patients with Asthma

Macrophages are the main cellular component in bronchoalveolar lavage fluid specimens obtained from normal patients. Other cell types, including T lymphocytes (CD3+) of both helper (CD4+) and suppressor-cytotoxic (CD8+) T-cell subsets, are also present, but the percentage of these cell types usually does not exceed 10%. Eosinophils, basophils and mast cells, and T lymphocytes have been found to be increased in bronchoalveolar lavage fluid samples in studies that have attempted to quantify the magnitude of the airway inflammatory response in patients with asthma [1, 2]. Recent findings suggest that T lymphocytes play a fundamental role in the induction of allergic inflammation. In fact, these cells not only recruit other specialized cells, such as eosinophils, by secreting interleukin-5 [1], they also promote local and systemic synthesis of IgE through the production of interleukin-4 [3, 4]. Lymphocytes with such functional activities are currently termed T-helper 2 (Th2), whereas the non-overlapping cell counterpart that secretes interferon- or interleukin-2 or both is termed T-helper 1 (Th1) and is involved in the delayed hypersensitivity immune reactions [3]. Although Th2-like activated T lymphocytes have been detected in the lungs of atopic asthmatic patients [5], it is still unclear whether these cells bear the or the less common T-cell receptor for antigen on their surface. This doubt is legitimated by the fact that many CD3+ intraepithelial lymphocytes found in the nasal mucosa of patients with allergic rhinitis are T cells that display the T-cell receptor heterodimer [6]. These findings prompted us to determine whether allergen-specific T lymphocytes are present in the bronchoalveolar lavage fluid of asthmatic patients and that, in these patients, they will be the major T-cell subset to disappear after systemic steroid treatment. Interestingly, our in vivo and in vitro studies provide evidence that apoptosis is the basic mechanism through which corticosteroid treatment acts on this T-lymphocyte type, thereby confirming the results of our previous investigations [7]. Methods Patients We studied 12 mildly symptomatic patients (6 children and 6 adults) with chronic asthma (FEV1 70% of that predicted for persons of their age and height) who were not receiving therapy with inhaled or oral corticosteroids, sodium cromoglycate, theophylline, or 2-agonists. Results of skin prick tests with purified Dermatophagoides pteronyssinus, D. farinae, and Parietaria judaica allergen extracts (Neo Abello, Madrid, Spain) were positive; results of an enzyme-linked immunosorbent assay (DPC Corporation, Los Angeles, California) for circulating allergen-specific IgE were also positive. None of the patients was a smoker or had had antecedent upper respiratory tract infection. Bronchoalveolar lavage fluid collection and fiberoptic bronchoscopy were done as previously described [8]. Ten healthy non-atopic, nonsmoking volunteers and patients diagnosed as having pulmonary sarcoidosis (n = 5) or extrinsic allergic alveolitis (n = 4), two pathologic conditions known to be associated with over-expanded lung CD4+ or CD8+ T-cell populations, were used as adult control groups. Age-matched uninfected children with cystic fibrosis (n = 5) or anatomic malformation (n = 4) of the airways served as controls for the cohort of asthmatic children. Bronchoalveolar lavage was repeated in 3 asthmatic patients after 1 week of therapy with deflazacort (Flantadin, Lepetit, Milano, Italy), 60 mg twice daily (a dose equivalent to 50 mg of prednisone), and then 3 weeks after therapy. We administered corticosteroids to these patients for exacerbation of their disease. We obtained informed consent from all study participants, and the clinical research was conducted in accordance with the local ethical committee guidelines. Lymphocyte Typing Pulmonary T cells were phenotyped with the following monoclonal antibodies: phycoerythrin-conjugated anti-CD3 (Ortho Pharmaceutical Corporation, Raritan, New Jersey), which recognizes up to 90% of T cells bearing the or T-cell receptor heterodimer; anti-CD4 and anti-CD8 (Ortho), which stain helper/inducer and cytotoxic T-cell subsets; and fluorescein-conjugated anti-TCR 1, anti-V1(a), and anti-V2(a) (T Cell Sciences, Cambridge, Massachusetts), which identify all T lymphocytes and the reciprocal subtypes expressing V1+ and V2+ gene products. In the analysis of two-color cytofluorimetric data (FACScan, Becton-Dickinson, Mountain View, California), we used the Lysis II program (Becton-Dickinson) to optimize gating of lymphocytes and to provide an objective means of excluding both debris and other cell types. T-Cell Enrichment In accordance with the manufacturers instructions (Dynal, A. S., Oslo, Norway), pulmonary mononuclear cells from 3 D. pteronyssinus-sensitive patients were enriched in TCR1-reactive T-cell subsets by negative magnetic immunoselection, using a mixture of anti- T-cell receptor monoclonal antibodies. This procedure yielded a + T-cell population of greater than 85%, which was used for culture and apoptotic cell death experiments. Proliferation Assay To assess the allergen-specificity of bronchoalveolar lavage T cells, 1.2 105 mononuclear cells and 4 104 -enriched lung T cells (the latter co-cultured with 8 104 autologous macrophages) from three D. pteronyssinus-sensitive patients and, because of the very small percentage of T cells in normal bronchoalveolar lavage fluid, 1.2 105 mononuclear cells from three controls were seeded in microplates and cultured in medium alone (RPMI-1640 [Gibco, Grand Island, New York] supplemented with fetal calf serum, L-glutamine, Hepes buffer, and antibiotics) or in the presence of 1 mug/mL purified D. pteronyssinus (Neo Abello, Madrid, Spain) or unrelated allergen Lolium perenne (purified L. perenne, Neo Abello) from the Graminaceae family. After 60 hours of culture, the cells were pulsed for 16 hours with 0.5 muCi [3H]-thymidine and were harvested, and the radioactivity was measured by liquid scintillation. Results are expressed as net cpm (counts per minute) [3H]-thymidine incorporation and reflect absolute cpm [3H]-thymidine uptake minus background cpm (cpm incorporated in the absence of allergen). Evaluation of Apoptotic Cell Death Apoptotic cells were qualitatively evaluated by agarose gel DNA electrophoresis [7] and quantitatively assessed as described previously [9]. Briefly, 1 106 TCR1+ pulmonary T cells were incubated for 24 hours with medium alone or dexamethasone (107 M), resuspended in 1.5 mL hypotonic propidium iodide solution and propidium iodide fluorescence of individual nuclei measured in a FACScan flow cytometer (Becton-Dickinson). We carried out control experiments using macrophage-depleted pulmonary T cells from healthy persons. Statistical Analysis Study populations were stratified according to age class. We used the Kruskall-Wallis one-way analysis of variance (with a significance level of 0.05) for statistical evaluation of the differences in bronchoalveolar lavage T-lymphocyte subset distributions. Pairwise comparisons for measuring the significance of the differences among mean values ( SE) calculated in the various groups (asthmatic patients compared with controls) were done with the Mann-Whitney U-test. In asthmatic patients (n = 12), the overall relation between serum IgE levels (IU/mL) and the percentages of pulmonary T cells was analyzed using the Spearman correlation test. The Statistical Package for the Social Sciences (SPS, Chicago, Illinois), version 4.0, was used for all statistical computations. Results As shown in Table 1, a significant proportion of CD3+ T lymphocytes in patient samples double-stained for the TCR1 monoclonal antibody, which defines + T lymphocytes. More than half of these T cells were CD4+, primarily of the V1+ subset, whereas the remainder of T cells found in the lung of our asthmatic patients were CD4 CD8, as shown by the fact that they never reacted with anti-CD8 monoclonal antibody. In addition, the percentage of T cells expressing the V2 isoform of the T-cell receptor was negligible in all samples tested (data not shown). Statistically significant differences were also obtained when the absolute numbers of pulmonary T cells from patients with asthma (mean count in adults, 29.6 2.5 104; in children, 23.2 3.1 104) were compared with those of the corresponding controls (mean count in adults, 0.2 0.1 104; P < 0.001; in children, 0.3 0.1 104; P < 0.001). Table 1. Bronchoalveolar Lavage T-Lymphocyte Subset Distribution in Patients with Allergic Asthma* The bronchoalveolar lavage T-cell concentration was not correlated with either specific serum IgE levels or any other clinical or functional (spirometric) variables (data not shown). In contrast, a relation was found between total serum IgE concentrations and pulmonary T-lymphocyte numbers, but this did not reach statistical significance (r = 0.21; P = 0.06). In the three in vitro experiments that we carried out, pulmonary T cells from D. pteronyssinus-sensitive patients proliferated in response to the specific allergen, with a net [3H]-thymidine incorporation ranging from 2400 to 4850 cpm, but did not proliferate in the presence of unrelated (L. perenne) allergen extract, with a [3H]-thymidine incorporation ranging from 120 to 450 cpm. Furthermore, lung T cells abruptly decreased and + CD4+ and CD8+ T lymphocytes increased in these patients after 1 week of treatment with glucocorticoids. Interestingly, the absolute numbers of T cells did not increase again when therapy was discontinued (Figure 1). The T-cell subpopulation was sensitive to steroid-induced programmed cell death in vitro. In fact, 24-hour dexamethasone incubation followed by flow-cytometric analysis with propidium iodide staining showed that the propidium iodide fluorescence profiles of apoptotic nuclei increased from 7% to 35% in dexamethasone-treated bronchoalveolar -enriched T cells, but not in control

CD30+ T Cells in Rheumatoid Synovitis: Mechanisms of Recruitment and Functional Role

High serum levels of soluble CD30 (sCD30) have been reported to better predict the response to second line therapy in rheumatoid arthritis (RA). It is believed that sCD30 is released by CD30+ T cells present in the RA synovium. However, both the mechanism of recruitment to the joint and the functional role of this T cell subset in the pathogenesis of the disease remain unknown. This study confirmed higher levels of sCD30 in the serum and synovial fluid (SF) of RA patients compared with normal controls. However, analysis of mRNA and cell surface CD30 expression showed that CD30+ T cells are detectable in the SF, but not in the synovial membrane. In contrast, T cells expressing the CD30 transcript, but not the surface molecule, were found in the peripheral blood of both RA and normal controls. CD30 surface expression was up-regulated by adhesion and migration through endothelium in vitro and in a delayed-type hypersensitivity model in vivo. Although the great majority of fresh or cloned CD30+ T cells from SF produced both IFN-γ and IL-4, CD30 expression strictly correlated with IL-4 synthesis in synovial T cell clones. In addition, CD30+ T cell clones also produced high amounts of the anti-inflammatory cytokine IL-10. On this basis, we would like to propose that synovial CD30+ cells may play a role in the control of the inflammatory response. Serum sCD30 may reflect such cell activity and, therefore, explain the previously demonstrated correlation between high sCD30 serum levels and positive response to therapy.

CD1-Restricted Recognition of Exogenous and Self-Lipid Antigens by Duodenal γδ+ T Lymphocytes

γδ T cells are present in the mucosal intestinal epithelia and secrete factors necessary to maintain tissue integrity. Ags recognized by these cells are poorly defined, although in mice non-classical MHC class I molecules have been implicated. Since MHC class I-like CD1 receptors are widely expressed at the surface of epithelial and dendritic intestinal cells and have the capacity to present lipid Ags to T cells, we hypothesized that these molecules might present autologous and/or exogenous phospholipids to intestinal γδ T lymphocytes. Intraepithelial T lymphocytes from normal human duodenal mucosal biopsies were cloned and exposed to natural and synthetic phospholipids using CD1a-, CD1b-, CD1c- or CD1d-transfected C1R lymphoblastoid or HeLa cell lines as APCs. Their cytolytic properties and regulatory cytokine secretion were also examined. Most clones obtained from duodenal mucosa (up to 70%) were TCRαβ+, and either CD4+ or CD8+, whereas 20% were CD4−CD8− (6 clones) or TCRγδ+ (12 clones). A relevant percentage (up to 66%) of TCRγδ+ but few (<5%) TCRαβ+ T cell clones responded to synthetic and/or natural phospholipids presented by CD1 molecules, as measured by both [3H]thymidine incorporation and IL-4 release assays. A Th1-like cytolytic and functional activity along with the ability to secrete regulatory cytokines was observed in most phospholipid-specific γδ T cell clones. Thus, a substantial percentage of TCRγδ+ but few TCRαβ+ from human duodenal mucosa recognize exogenous phospholipids in a CD1-restricted fashion. This adaptive response could contribute to mucosal homeostasis, but could also favor the emergence of inflammatory or allergic intestinal diseases.

Identification of regulatory T cells in systemic lupus erythematosus

The concept that regulatory T cells (Treg) play a key role in both development and maintenance of autoimmune response in rheumatic diseases is well accepted. In recent years, several studies analyzed Treg cell phenotype and function in systemic lupus erythematosus (SLE), the prototypical systemic autoimmune disorder in humans. Although qualitative and/or quantitative abnormalities of Treg cells have been shown, data are often conflicting. This may depend on the selection of patients with different degrees of disease activity or on immunosuppressive treatments that can alter Treg cell findings. Among several proposed surface or intracellular Treg cell markers, CD25 at high level of expression and the transcription factor Foxp3 are the two most investigated in SLE. Despite the glucocorticoid-induced TNF receptor-related protein (GITR) represents a reliable phenotypic marker of murine Treg cells, little is known about its role in humans, in particular in the course of systemic autoimmune disorders. Preliminary data seems to suggest that this marker may represent a good tool to identify cell populations included within Treg cell subsets.