Marco De Carli

Human CD4+ T cell clones produce and release nerve growth factor and express high-affinity nerve growth factor receptors

BACKGROUND Increasing evidence shows that nerve growth factor (NGF) plays a role in the complex and fascinating linkage between the nervous and the immune systems due to its ability to modulate functions of several inflammatory cells. OBJECTIVE To investigate NGF receptor expression and NGF production and release by human CD4+ cells clones, which have primary relevance in modulating inflammatory events through their different subsets of functional phenotypes. METHODS The expression of NGF and a transmembrane tyrosine kinase (TrkA) was evaluated by immunohistochemistry and flow cytometry analysis in five T(H0), six T(H1), and five T(H2) cell clones derived from human circulating mononuclear blood cells. Moreover, the amount of NGF protein was assessed by measuring the NGF levels in culture supernatants of the T cell clones before stimulation and 48 hours after phytohemagglutinin (PHA) activation by use of an immunoenzymatic assay. RESULTS Our data have shown that in unstimulated conditions, human CD4+ T cell clones express both immunoreactivity for NGF and the TrkA NGF receptor irrespective of their cytokine profile. Moreover, T(H1) and T(H2) clones, but not T(H0) clones, secrete NGF in basal conditions. PHA activation induces NGF secretion in T(H0) clones and a significant increase of NGF levels in T(H2) (p < 0.05), but not in T(H1) culture supernatants. CONCLUSIONS Results obtained represent the first evidence of TrkA expression and NGF production and release in human CD4+ cell clones and suggest a possible functional role of NGF in modulating the immune and inflammatory network.

Human Th1 and Th2 cells: functional properties, regulation of development and role in autoimmunity.

Evidence has accumulated suggesting the existence in humans of polarized T helper (Th) cell subsets, coded as Th1 and Th2, with defined cytokine secretion profiles. Immune responses to intracellular bacteria and viruses result in the preferential development of the Th1 cell subset. Th1 cells express cytolytic activity against antigen-presenting cells and provide helper function for IgM, IgG and IgA synthesis only at low T/B cell ratios. In contrast, Th2 cells develop in response to allergens or helminth antigens, provide help for all immunoglobulin classes, including IgE, and lack cytolytic potential. The cytokine milieu in the microenvironment plays a fundamental role in determining the functional phenotype of the subsequent antigen-specific Th1 or Th2 responses. In recent years it has become clear that Th1 and Th2 cells play different roles not only in protection against exogenous offending agents, but also immunopathology. Th2 cells are involved in immunopathology induced by helminths and are responsible for the initiation and maintenance of allergic disorders. Th1 cells seem to be involved in contact dermatitis, acute allograft rejection and organ-specific autoimmunity, such as thyroid autoimmune disorders, diabetes mellitus or multiple sclerosis, whereas less polarized patterns of Th cells are detectable in target organs of patients with rheumatoid arthritis. Sjogrens syndrome or systemic lupus erythematosus.

Oxidative stress stimulates IL-4 and IL-6 production in mast cells by an APE/Ref-1-dependent pathway

Mast cells are exposed to an oxidative environment in the course of allergic and inflammatory reactions. We have examined the effects of H2O2 stimulation in a primary rat basophilic leukemia cell line (RBL‐2H3) and compared with IgE‐dependent stimulation. Like IgE stimulation, H2O2 up‐regulates IL‐4 and IL‐6 gene expression and cytokine secretion, shows a little effect on IL‐5 but does not induce IL‐10 gene expression. Simultaneous H2O2 treatment and FcϵRI triggering of mast cells has additive effects on IL‐4 expression. In addition, we show that both stimuli induce the nuclear translocation of APE/Ref‐1, a bifunctional enzyme that stimulates the DNA‐binding activity of several transcription factors through the reduction of highly reactive cysteines. Conditional inactivation of APE/Ref‐1 expression abolishes H2O2‐induced IL‐4 and IL‐6 gene expression but does not affect that induced by FcϵRI stimulation. Our findings indicate that oxidative stress activates the gene expression of a specific cytokine pattern in mast cells through an APE/Ref‐1‐dependent pathway, which is distinct from theone that is activated by FcϵRI stimulation. Nonetheless, H2O2 and FcϵRI signalings are additive in augmenting IL‐4 production. Most importantly, oxidative stress can induce a pro‐type 2 inflammatory response from mast cells that is independent of FcϵRI stimulation.

The mast cell: an antenna of the microenvironment that directs the immune response

Mast cells (MCs) have long been considered as critical effector cells during immunoglobulin (Ig)E‐mediated allergic disease and immune response to parasites. Recent studies, however, suggest that this understanding of MC function is incomplete and does not consider the complex roles that MCs play in adaptive and innate immunity. The added function gives an innovative vision of regulation of immune responses and the development of autoimmune diseases. It had been assumed that the aggregation of Fc ɛ receptor I with IgE and specific antigen is the main stimulus able to induce the MC activation, degranulation, release, and generation of mediators of the allergic reaction. However, MCs exhibit an array of molecules involved in cell–cell and cell–extracellular matrix adhesion, mediating delivery of costimulatory signals that empower those cells with an ability to react to multiple nonspecific and specific stimuli. Their tissue distribution and their capability to release many cytokines after stimulation indicate MCs as potential regulatory linkers between innate and acquired immunity. In this review, we will summarize some findings on the roles of MCs in innate and acquired immunity, on the molecular mechanism and signaling pathways, and on selective signals that induce discrete MC response and its ability to polarize adaptive‐immune response.

In vivo relevance of CD30 in atopic dermatitis.

CD30 expression was evaluated by immunohistochemistry in lesional skin biopsies of eight patients with active atopic dermatitis (AD) and three patients with allergic contact (nickel‐induced) dermatitis (ACD). CD30 expression was also assessed in a large panel of CD4 + and CDS + T‐cell clones generated from the skin biopsies of four patients with AD. Finally, the levels of soluble CD30 (sCD30) were measured in the serum of 41 patients with AD, 19 patients with ACD, and 60 healthy controls. In all specimens of lesional AD skin, where the great majority of infiltrating cells were CD4+ T cells, remarkable numbers of cells were CD30+, whereas virtually no CD30 + cells were found in the skin of patients with ACD. In CD4+ T‐cell clones generated from the lesional AD skin, most of which produced both interleukin (IL)‐4 and interferon‐gamma (IFN‐γ) (Th0–like cells) or IL‐4 and 1L‐5, but not IFN‐γ (Th2–like cells), CD30 expression directly correlated with the ability to produce IL‐4 and IL‐5, but was inversely related to IFN‐γ production. High levels of sCD30 (correlated with disease activity: r= 0.618) were detected in the serum of most AD patients, whereas there was no increase of sCD30 levels in the serum of patients with ACD. These data support the view that Th0/Th2–type responses predominate in the skin of patients with AD and suggest that the presence of CD30 + T cells in tissues and/or increased levels of sCD30 in biologic fluids are indicative of Th2–dominated responses.

No relationship between skin-infiltrating TH2-like cells and allergen-specific IgE response in atopic dermatitis.

More than 500 CD4+ T-cell clones (TCCs) derived from the skin of eight patients with atopic dermatitis (AD), two patients with nonatopic dermatologic disorders, two patients with allergic rhinitis, and one healthy nonatopic donor were analyzed for both their pattern of cytokine production and their antigen specificity. The proportions of TCCs from patients with AD producing interleukin-4 in response to stimulation with phorbol 12-myristate 13-acetate plus anti-CD3 antibody were higher, whereas the proportions of interferon-gamma--producing TCCs were lower than those of control subjects. In two patients with AD, the majority of TCCs had a TH2/TH0-like phenotype, whereas in six patients with AD a TH1/TH0-like phenotype was prevalent. TCCs with a TH2/TH0-like phenotype were also isolated from the healthy skin of two patients with allergic rhinitis and one nonatopic donor. In contrast, no TH2-like TCCs were derived from the skin of the two patients with dermatologic disorders of nonallergic origin. No unambiguous correlations was found between the proportions of TCCs producing interleukin-4 or interferon-gamma (or of TCCs with TH2- or TH1-like profile) and the level of total serum IgE, suggesting that CD4+ T cells infiltrating the atopic skin do not play a major role in the production of serum IgE antibodies. When TCCs from five patients with AD were examined for their specificity, the proportions of allergen-specific (Dermatophagoides pteronyssinus and Lol p 1) clones were consistently 6% or lower even in patients with high titers of ryegrassor D. pteronyssinus-specific IgE antibodies. Because similar percentages of allergen-specific TCCs were found in skin from two healthy control subjects, the role of aeroallergens in favoring and maintaining skin lesions in patients with AD remains unclear.

The IgE response in atopy and infections

The IgE antibody system represents an important defense mechanism against offending agents coming from the respiratory and gastrointestinal tracts. The human pathological condition most commonly associated with hyperproduction of IgE is atopy, the familial allergic disorder of immediate-type hypersensitivity to environmental allergens. Dysregulation of IgE synthesis has also been observed in clinical disorders often associated with immunodeficiency and/or infections, such as the Hyper-lgE syndromes (1), parasitic infestations, some primary and secondary immunodeficiencies (2, 3), AIDS (4), the acute phase of GVHD (5). Therefore, the understanding of mechanisms involved in IgE regulation is of primary importance in order to define the alterations responsible for allergic diseases, as well as other pathological conditions characterized by hyperproduction of IgE. In the first part of this paper we have summarized the mechanisms that regulate the synthesis of IgE in humans. In the second part, we discuss the pathophysiological aspects of IgE dysregulation in disorders characterized by hyperproduction of IgE.

Co-Occurrence of Chronic Spontaneous Urticaria with Immunoglobulin A Deficiency and Autoimmune Diseases

Background: Immunoglobulin (Ig) A deficiency is a primary immunodeficiency in which autoimmunity is frequently observed. Thirty to fifty percent of patients with spontaneous chronic urticaria have autoantibodies that are able to cross-link FcεRI on mast cells and basophils. Methods: We investigated whether spontaneous chronic urticaria in patients with IgA deficiency meets the criteria for autoimmunity. Four patients were screened for positivity to a skin prick test and an autologous serum skin test and for the presence of other autoimmune diseases. Patient sera were tested for the ability to activate basophils and mast cells in vitro by measuring surface CD63 expression and β-hexosaminidase release, respectively. Results: The autologous serum test was positive in all patients, and patient sera were found to induce CD63 upregulation on basophils and degranulation of an LAD2 mast cell line. Moreover, all patients were affected by other autoimmune disorders. Conclusion: For the first time, these data point out chronic autoimmune urticaria in subjects with an IgA deficiency and confirm that different autoimmune disorders are common among patients with an IgA deficiency. Patients with chronic autoimmune spontaneous urticaria should be screened for IgA deficiency, especially if they are affected by other autoimmune disorders. Thus, spontaneous urticaria could mirror more complex systemic diseases, such as immune deficiency.

Molecular targets of atypical antipsychotics: From mechanism of action to clinical differences

ABSTRACT The introduction of atypical antipsychotics (AAPs) since the discovery of its prototypical drug clozapine has been a revolutionary pharmacological step for treating psychotic patients as these allow a significant recovery not only in terms of hospitalization and reduction in symptoms severity, but also in terms of safety, socialization and better rehabilitation in the society. Regarding the mechanism of action, AAPs are weak D2 receptor antagonists and they act beyond D2 antagonism, involving other receptor targets which regulate dopamine and other neurotransmitters. Consequently, AAPs present a significant reduction of deleterious side effects like parkinsonism, hyperprolactinemia, apathy and anhedonia, which are all linked to the strong blockade of D2 receptors. This review revisits previous and current findings within the class of AAPs and highlights the differences in terms of receptor properties and clinical activities among them. Furthermore, we propose a continuum spectrum of “atypia” that begins with risperidone (the least atypical) to clozapine (the most atypical), while all the other AAPs fall within the extremes of this spectrum. Clozapine is still considered the gold standard in refractory schizophrenia and in psychoses present in Parkinsons disease, though it has been associated with adverse effects like agranulocytosis (0.7%) and weight gain, pushing the scientific community to find new drugs as effective as clozapine, but devoid of its side effects. To achieve this, it is therefore imperative to characterize and compare in depth the very complex molecular profile of AAPs. We also introduce relatively new concepts like biased agonism, receptor dimerization and neurogenesis to identify better the old and new hallmarks of “atypia”. Finally, a detailed confrontation of clinical differences among the AAPs is presented, especially in relation to their molecular targets, and new means like therapeutic drug monitoring are also proposed to improve the effectiveness of AAPs in clinical practice.

T Lymphocytes from tonsil and peripheral blood show different cytolytic and helper activities

T lymphocytes from tonsil (To) and peripheral blood (PB) of 4 tonsillectomized children were subjected to clonal expansion with PHA in order to analyze at single cell level their cytolytic activity and their ability to produce interleukins such as IL-2, IFN-gamma and IL-4. Analyzing all T-cell clones (CD4+ and CD8+) obtained from To in comparison with those from PB, a reduced proportion of cells with lectin-dependent cytolytic activity (LDCC) (25% vs. 42%, p less than 0.01) and natural killer (NK) activity (18% vs. 31%, p less than 0.02) was found. These differences were proportionally related to the lower number of CD8+ T-cells in To than in PB. The proportion of CD4+ clones able to produce IL-2 and/or IL-4 were higher in To (75% and 61%) than in PB (52% and 25%, p less than 0.001 and p less than 0.001, respectively). In contrast, the proportion of CD4+ clones able to produce IFN-gamma was similar (53% and 58%) in both series of clones. According to the patterns of lymphokine synthesis, tonsillar T-cells differed from PB T-cells as follows: 1) the number of Th1-like CD4+ clones producing IL-2 and/or IFN-gamma (but not IL-4) were 23% vs. 44% in PB (p less than 0.001); 2) there was no difference between To and PB in the proportion of CD4+ clones producing IL-4 alone (Th2 clones: 9% vs. 8%); 3) CD4+ clones synthesizing IL-2, IL-4 and IFN-gamma at the same time were more frequent in To than in PB (Th3 clones: 53% vs. 17%, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)