Complementary roles for lipid and protein allergens in triggering innate and adaptive immune systems.

BACKGROUND: Recent advances in allergy research mostly focussed on two major headings: improving protein allergen purification, which is aimed towards a better characterization of IgE- and T-cell reactive epitopes, and the potential new role for unconventional innate and regulatory T cells in controlling airway inflammation. These advancements could appear to be in conflict each other, as innate T cells have a poorly-defined antigen specificity that is often directed toward nonprotein substances, such as lipids. METHOD: To reconcile these contrasting findings, the model of cypress pollinosis as paradigmatic for studying allergic diseases in adults is suggested. RESULTS: The biochemical characterization of major native protein allergens from undenatured pollen grain demonstrated that the most relevant substance with IgE-binding activity is a glycohydrolase enzyme, which easily denaturizes in stored grains. Moreover, lipids from the pollen membrane are implicated in early pollen grain capture and recognition by CD1(+) dendritic cells (DC) and CD1-restricted T lymphocytes. These T cells display Th0/Th2 functional activity and are also able to produce regulatory cytokines, such as IL-10 and TGF-beta. CD1(+) immature DCs expand in the respiratory mucosa of allergic subjects and are able to process both proteins and lipids. CONCLUSION: A final scenario may suggest that expansion and functional activation of CD1(+) DCs is a key step for mounting a Th0/Th2-deviated immune response, and that such innate response does not confer long-lasting protective immunity.

Biological effects of montelukast, a cysteinyl-leukotriene receptor-antagonist, on T lymphocytes.

BACKGROUND: Montelukast (MNT), a cysteinyl-leukotriene receptor (Cys-LTR) antagonist, has anti-inflammatory activity in the treatment of allergic diseases. If this effect is due only to blocking leukotrienes or also owing to inhibiting proliferation and survival of inflammatory cells, is actually unknown. OBJECTIVE: Testing the hypothesis that MNT could influence T lymphocyte functional behaviour in vitro. METHODS: Normal T lymphocytes were analysed for surface expression of Cys-LTR(1) and Cys-LTR(2) by means of monoclonal antibodies (mAbs), in the resting state and after activation with T helper type 2 cytokine or T cell receptor (TcR) stimulation. Proliferative activity, as well as IL-4 andIFN-gamma production, were simultaneously determined in samples exposed to molar concentrations of MNT from 10(-8) to 10(-5). Programmed cell death in cultured samples was evaluated by means of propidium iodide and fluorescein isothiocyanate-conjugated anti-Annexin V mAb staining. The complementary DNA microarray technique was adopted to identify gene products involved in apoptosis induction. RESULTS: Resting T cells expressed low levels of Cys-LTR. Upon anti-CD3 mAb activation, a progressive increase in Cys-LTR(1) and -LTR(2) expression was observed. Exposure to MNT reduced proliferative response to TcR engagement, increased IFN-gamma production and led to apoptosis at minimal concentrations of 10(-6) M. A progressive loss in BAD and B cell lymphoma/leukaemia-2 activities, and an increase in the expression of CD27, TRAF3, TRAIL, p53 and Fas genes were also observed. CONCLUSIONS: Biological effects of MNT delineate a complex picture of gene activation and repression, probably induced by Cys-LTR blockade. The induction of apoptosis in allergen-specific T cell population, as a final result, appears fundamental in the treatment of asthma.

Flow cytometric measurement of intracellular IFN-gamma induction in aged subjects before and after parenteral influenza vaccination.

Flow cytometric analysis, used to study intracellular expression of IFN-gamma in peripheral blood mononuclear cells (PBMC) from aged volunteers before and after parenteral influenza vaccination, was found capable of rapidly detecting influenza antigen induced variation of IFN-gamma expression. Although the vaccine was capable of generating a satisfactory antibody response, it did not stimulate an increase in the percentage of IFN-gamma positive cells.

In vivo activated T cells in rheumatoid synovitis. Analysis of Th1- and Th2-type cytokine production at clonal level in different stages of disease.

T-cell cytokines play a crucial role in the pathogenesis and progression of rheumatoid arthritis (RA). Their detection in the joint, however, is impaired by the complex network present in the synovium. Although many synovial T cells show signs of previous activation, only a few express interleukin (IL)-2 receptor, marker of recent activation. The aim of this study was to analyse the cytokine production by in vivo activated (IL-2R +) T cells from RA at different stages of the disease. For this purpose, T cells were isolated from peripheral blood and synovial fluid of four patients with active RA, two at the onset of the disease, one in the early phase during treatment, one in long-lasting chronic phase. One patient was studied at the onset of the disease and 52 months later. Cells were initially expanded with a low dose of IL-2, cloned and analysed for cytokine production. The results showed a strong predominance of T helper (Th) 1 clones in the blood and a slight prevalence of Th0 clones in the joint of all the four patients. Interferon-gamma and IL-2 production was higher in the long-lasting RA, whereas IL-4 synthesis was prevalent in early RA. Enrichment in IL-10-producing clones was present only in the joint of the untreated patients. The longitudinal study confirmed the differences in cytokine production between early and late phases of disease. These data confirm that RA is mainly a Th1-driven condition. However, in vivo activated synovial T cells produce also Th2-type anti-inflammatory cytokines, such as IL-4 and IL-10. The synthesis of both cytokines is a feature of the very early phase of RA, although the selective recruitment of IL-10-producing T cells is quickly lost.

CD4+IL13+ T lymphocytes at birth and the development of wheezing and/or asthma during the 1st year of life.

BACKGROUND: Despite our knowledge that maternal inheritance influences the development of asthma in childhood, attempts to identify a clear-cut Th2-oriented cytokine production by T lymphocytes at birth have given conflicting results. The prognostic significance of these cells for asthma development later in life remains to be determined. METHODS: We evaluated at the single cell level Th1- and Th2-type cytokines in 208 randomly selected cord blood mononuclear cell (CBMC) samples obtained from pregnant women (group A, n = 68 with diagnosed respiratory allergic disease; group B, n = 140, with no evidence of atopy), and prospectively followed newborns for 1 year. RESULTS: There was no difference in IFN-gamma, IL-4 and IL-5 production at birth between both groups, whereas a correlation between CD4+IL13+ lymphocytes from CBMC samples derived from atopic mothers and the occurrence of wheezing and/or asthma during the 1st year of life was found. CONCLUSIONS: Our observations suggest that the intracellular cytokine profile of cord blood CD4+ cells, in terms of IL-13 production, could be considered a useful tool for a more accurate identification of newborns from atopic mothers who are at high risk of developing asthma.

Neutrophil peripheral count and human leukocyte elastase during chronic lithium carbonate therapy.

Plasma levels of human polymorphonuclear elastase (PMN-E) are considered a marker of granulocyte activation and can potentially complement the peripheral neutrophil count in laboratory and pathophysiological settings. Neutrophilic leukocytosis is a well known effect of lithium therapy, but there is no information about the concomitant behaviour of PMN-E in these patients. The aim of this study was to evaluate both polymorphonuclear leukocyte count and plasma PMN-E levels in depression patients undergoing chronic lithium therapy. Absolute and differential leukocyte count in venous peripheral blood was determined by an automated method, and PMN-E evaluated by enzyme immunoassay. 39 patients (11 males, 28 females; mean age 43. +/- 6.02) with depression disorders were studied, during lithium carbonate therapy. Neutrophilia (neutrophil count > 7.500x10(9) cells per liter) was found in 7 (18%) patients and an increase in plasma PMN-E levels (PMN-E > 56 microg per liter ) in 6 (15%). No correlations were found between neutrophil count, plasma concentration of PMN-E, plasma level of lithium and duration of therapy. The results show that in these patients, not only the PMN count but also elastase levels can exceed the normal range. The absence of correlation between these two parameters suggests that the state of PMN activation is not linked to their number in peripheral blood.

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-gamma 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.

Role of T-helper type 2 cytokines in down-modulation of fas mRNA and receptor on the surface of activated CD4(+) T cells: molecular basis for the persistence of the allergic immune response.

The mechanisms responsible for persistence of T lymphocytes at the sites of allergic inflammation are not completely understood. Activated T cells, usually expressing Fas on their surface, undergo activation-induced apoptotic death, thus limiting the dangerous consequences of a persistent immune reaction. We have previously shown that pulmonary T lymphocytes from untreated asthmatic subjects do not express surface Fas receptors nor do they contain Fas mRNA, yet they display normal levels of Fas ligand. This is not an inherited defect and is confined to mucosal T cells. To gain insights into the mechanism responsible for these findings, we performed a set of experiments with both purified Dermatophagoides pteronyssinus allergen and recombinant human cytokines: interleukin 2 (IL-2), IL-4, IL-5, transforming growth factor beta1, interferon gamma, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In vitro exposure of purified CD4(+) lymphocytes to allergen yielded only transient up-regulation of surface Fas but did not influence susceptibility to Fas-mediated cell death. T-helper type 2 cytokines (IL-4, IL-5, and GM-CSF) had a dose-dependent and specific inhibitory effect on Fas mRNA, suggesting a new fundamental biological role in the survival of inflammatory cells during allergen exposure.

Expression of B7 co-stimulatory molecules and CD1a antigen by alveolar macrophages in allergic bronchial asthma.

BACKGROUND: Lung allergen recognition that takes place in the airways of asthmatic subjects is still a controversial matter. OBJECTIVE: We hypothesized that a rapid allergen recognition process requires the presence, at the mucosal surface, of professional APC, such as B7+ alveolar macrophages (AM) and/or CD1+ dendritic cells, which usually have a lower expression in the normal human lung. METHODS: Studies were performed on bronchoalveolar lavage (BAL) fluid collected from 10 untreated allergic subjects and 10 adult normal volunteers. Further controls consisted of five untreated pulmonary sarcoidosis (PS) and four extrinsic allergic alveolitis (EAA) individuals. To ascertain whether T helper 2-type cytokines or allergen influence B7 and CD1 antigen expression, in vitro studies were carried out using unprimed (naive) cord blood plastic-adherent monocytes. RESULTS: Cytofluorymetric analysis revealed that AM from asthmatics, unlike those from normal subjects or patients with PS or EAA, overexpressed B7-2, CD1a and, to a lesser extent, B7-1 surface molecules. Immunohistochemical studies confirmed the presence of CD1+ dendritic cells in the BAL fluid from asthmatic subjects. On in vitro cultured naive cord blood monocytes both purified Dermatophagoides pteronyssinus allergen and T-cell cytokines, i.e. IL-4 and granulocyte macrophage colony-stimulating factor, induced surface expression of B7-2 and CD1a receptors, whereas they had no appreciable effect on that of B7-1 membrane molecule. CONCLUSIONS: Taken together, these findings support the proposal that airways of atopic individuals are equipped with professional APC that synergize with allergen-specific T cells for the recognition of intact allergens. When the recognition process takes place, asthmatic symptoms could develop in genetically susceptible individuals.

Defective expression of Fas messenger RNA and Fas receptor on pulmonary T cells from patients with asthma.

BACKGROUND: Inflammation at sites of target organs seems to be the pathologic hallmark of respiratory allergic diseases, but why this response cannot be turned off in atopic persons is not known. Programmed cell death (apoptosis) mediated by Fas/APO-1 (CD95), a 45-kD surface protein belonging to the tumor necrosis factor receptor family, is important in the resolution of all inflammatory immune responses. OBJECTIVE: To test whether the expression of Fas receptor is defective in allergen-specific pulmonary T lymphocytes from persons with asthma. DESIGN: 12-month prospective study. SETTING: University allergy and immunology clinic. PATIENTS: 12 untreated persons with newly diagnosed allergic asthma who underwent bronchoalveolar lavage. Ten normal persons served as controls. MEASUREMENTS: Fas receptor expression was studied by using surface double-color cytofluorometry on pulmonary and circulating T lymphocytes. Fas messenger RNA (mRNA) was searched for in bronchoalveolar lavage cells from patients and controls by reverse transcription polymerase chain reaction (PCR). In vitro induction of DNA fragmentation, as an expression of cell death induced by an IgM anti-Fas monoclonal antibody, was assessed by propidium iodide staining and agarose gel electrophoresis. In vitro modulation of surface Fas receptor was studied on pulmonary T lymphocytes stimulated with anti-CD3 monoclonal antibody and interleukin-2 or interleukin-4. RESULTS: Pulmonary T lymphocytes from patients as opposed to controls did not undergo DNA fragmentation after in vitro exposure to IgM anti-Fas. Other activation markers (CD25, HLA-DR, and CD45R0) were displayed, but surface Fas expression was always negative. A remarkable proportion of T cells from controls showed a clear double-staining pattern. Reverse transcription PCR for Fas mRNA yielded the same results. Circulating T lymphocytes from patients and controls included similar percentages of CD3+ Fas+ cells. Pulmonary T cells from both patients and controls showed upregulation of Fas receptor expression after in vitro anti-CD3 stimulation; co-culturing with interleukin-4 downmodulated surface Fas receptor expression on T cells from patients; it was less effective in controls. CONCLUSIONS: Hypoexpression of Fas mRNA and surface Fas receptor on pulmonary CD3+ T lymphocytes may explain the persistence of inflammatory cellular infiltrates in allergic bronchial asthma.