Anne Tsicopoulos

Increased secretion of tumor necrosis factor α and interleukin-6 by alveolar macrophages consecutive to the development of the late asthmatic reaction

The late asthmatic reaction (LAR), consecutive to bronchial allergen challenge, is characterized both by the influx of various cells in proximal and distal airways and by the enhancement of bronchial hyperresponsiveness. However, the exact conditions for the development of the inflammatory reaction during the LAR remain to be specified. Since monokines play a key role in inflammatory processes, particularly in the lung, the production of tumor necrosis factor-alpha (TNF-alpha), interleukin; 1-beta (IL-1-beta) and interleukin-6 (IL-6) by alveolar macrophages (AM), collected 18 to 20 hours after exposure to allergen, was evaluated in 15 allergic subjects with asthma submitted to a challenge test with Dermatophagoides pteronyssinus (N = 6) or with wheat flour (N = 9) and in three healthy subjects. After bronchial provocation test, four patients presented no bronchial response (group 1), and six patients, a single early reaction (group 2). In contrast, five patients developed successively an immediate plus a late response (group 3). The monokine production was compared to that from nine allergic subjects with asthma studied at baseline (group 0) and from 11 unchallenged healthy subjects (control subjects). Measurements of cytokines were evaluated for TNF-alpha and IL-1-beta by a specific immunoradiometric assay, whereas IL-6 levels were appreciated by the proliferation of 7TD1 cells. No detectable amounts of TNF-alpha, IL-1-beta, and IL-6 were in bronchial alveolar lavage fluid, even after a tenfold concentration. In contrast, a significant increase of TNF-alpha (10,642 +/- 3127 U/ml) and IL-6 (1250 +/- 427 U/ml) concentrations was noted in AM supernatants from patients exhibiting an LAR (group 3) compared to cells recovered from groups 2, 1, and 0 and to challenged or unchallenged control subjects (805 +/- 244, 995 +/- 521, 1269 +/- 524, 688 +/- 85, and 445 +/- 74 pg of TNF-alpha per milliliter, respectively; 190 +/- 64, 114 +/- 91, 242 +/- 95, 80 +/- 9, and 54 +/- 19 U/ml of IL-6 per milliliter, respectively). No modification of IL-1-beta contents could be detected between the different groups. A significant correlation was detected between concentrations of TNF and IL-6 (r = 0.92; p less than 0.001). These results demonstrate TNF-alpha and IL-6 secretion by AM consecutively to the development of LAR in allergic subjects with asthma, confirming that AMs are activated after allergen challenge.(ABSTRACT TRUNCATED AT 400 WORDS)

IL-9 and its receptor in allergic and nonallergic lung disease: Increased expression in asthma

BACKGROUND Bronchial asthma is a chronic inflammatory disease associated with genetic components. Recently IL-9 has been reported as a candidate gene for asthma and to be associated with bronchial hyperresponsiveness and elevated levels of total serum IgE. OBJECTIVE To investigate the contribution of IL-9 to the pathogenesis of asthma, we examined the expression of IL-9 and its receptor (IL-9R) in bronchial tissue from subjects with atopic asthma (n = 10), chronic bronchitis (n = 11), and sarcoidosis (n = 9) and from atopic (n = 7) and nonatopic (n = 10) healthy control subjects. METHODS Bronchial biopsy specimens were examined for the presence of IL-9 and IL-9R protein and messenger RNA (mRNA) by immunocytochemistry and in situ hybridization, respectively. To phenotype the cells expressing IL-9 in asthmatic tissue, combined in situ hybridization and immunocytochemistry was also performed. RESULTS There was a highly significant difference (P <.001) in the expression of IL-9 mRNA in asthmatic airways (20.6 +/- 4.0 cells/mm of basement membrane) compared with chronic bronchitis (5.6 +/- 4.4), sarcoidosis (2.5 +/- 1.8), atopic control subjects (7.7 +/- 2.2), and healthy control subjects (2.7 +/- 2.3). The number of IL-9 immunoreactive cells was also greater in asthmatic patients compared with the other groups (P <.05). Although the level of IL-9R mRNA expression did not differ in any of the groups (P >.05), IL-9R immunoreactivity was significantly higher in asthmatic compared with control subjects. Furthermore, IL-9 mRNA expression levels were also significantly correlated with FEV(1) (P <.05) and the airway responsiveness to methacholine producing a 20% fall in FEV(1) (P <. 01). The cells expressing IL-9 mRNA in asthmatic tissue were CD3(+) lymphocytes (68%), major basic protein(+) eosinophils (16%), and elastase(+) neutrophils (8%). CONCLUSION The results of this study demonstrate the potential of IL-9 to be a marker for atopic asthma and furthermore suggest an important role for this cytokine in the pathophysiologic mechanisms of this disease.

Human Endothelial-Cell Specific Molecule-1 Binds Directly to the Integrin CD11a/CD18 (LFA-1) and Blocks Binding to Intercellular Adhesion Molecule-1

ICAMs are ligands for LFA-1, a major integrin of mononuclear cells involved in the immune and inflammatory processes. We previously showed that endothelial cell specific molecule-1 (ESM-1) is a proteoglycan secreted by endothelial cells under the control of inflammatory cytokines. Here, we demonstrate that ESM-1 binds directly to LFA-1 onto the cell surface of human blood lymphocytes, monocytes, and Jurkat cells. The binding of ESM-1 was equally dependent on Ca2+, Mg2+, or Mn2+ divalent ions, which are specific, saturable, and sensitive to temperature. An anti-CD11a mAb or PMA induced a transient increase in binding, peaking 5 min after activation. Direct binding of ESM-1 to LFA-1 integrin was demonstrated by specific coimmunoprecipitation by CD11a and CD18 mAbs. A cell-free system using a Biacore biosensor confirmed that ESM-1 and LFA-1 dynamically interacted in real time with high affinity (Kd = 18.7 nM). ESM-1 consistently inhibited the specific binding of soluble ICAM-1 to Jurkat cells in a dose-dependent manner. These results suggest that ESM-1 and ICAM-1 interact with LFA-1 on binding sites very close to but distinct from the I domain of CD11a. Through this mechanism, ESM-1 could be implicated in the regulation of the LFA-1/ICAM-1 pathway and may therefore influence both the recruitment of circulating lymphocytes to inflammatory sites and LFA-1-dependent leukocyte adhesion and activation.

Release of granule proteins by eosinophils from allergic and nonallergic patients with eosinophilia on immunoglobulin-dependent activation

The release of eosinophil peroxidase (EPO) and eosinophil cationic protein (ECP) was evaluated after incubation of eosinophils (EOSs) from allergic subjects with the specific allergen or with anti-IgE monoclonal antibodies (MAbs). High levels of EPO could be released after addition of the specific allergen (and not unrelated ones) or anti-IgE MAb. Moreover, EPO release with the two stimuli was significantly correlated both in allergic and in nonallergic patients. In the same supernatants, another granule protein, ECP, could not be detected, suggesting a lack of correlation between EPO and ECP release after IgE-dependent stimulation. However, when EOSs with surface-IgA antibodies were incubated with anti-IgA MAb, both EPO and ECP were released. In contrast, incubation of EOSs with anti-IgG MAb induced mainly the release of ECP and not EPO. These results indicate that pharmacologically active mediators can be released by EOSs from allergic and nonallergic patients on immunoglobulin-dependent activation. The results also confirm the hypothesis of a selective release of the various granule proteins and raise the question of transduction signals delivered by the three Fc receptors (Fc epsilon R, FC alpha R, and FC gamma R) present on human EOSs.

Kinetics of cell infiltration and cytokine messenger RNA expression after intradermal challenge with allergen and tuberculin in the same atopic individuals

BACKGROUND Previous studies, in which one time point was used, have shown that cells infiltrating skin biopsy specimens taken during allergen-induced late-phase responses (LPR) had a TH2-like (interleukin-4 [IL]-4 and IL-5 mRNA+) cytokine profile, whereas in delayed-type hypersensitivity (DTH) there was a predominant TH1-type pattern. OBJECTIVE The study was designed to examine the kinetics of accumulation of inflammatory cells and cells expressing mRNA for TH2- or TH1-type cytokines in LPR and DTH elicited simultaneously in the same subjects. METHODS Immunocytochemistry (alkaline phosphatase anti-alkaline phosphatase technique) and in situ hybridization were used to analyze skin biopsy specimens taken during allergen-induced LPR. RESULTS In LPR elevated numbers of CD3+ and CD4+ cells, eosinophils, neutrophils, and IL-4 and IL-5 mRNA+ cells were detected as early as 1 hour after allergen challenge, with a peak at 6 hours, which was maintained for up to 96 hours. A small but significant delayed increase in macrophages, CD8+ and CD25+ cells, and IL-2 and interferon-gamma mRNA+ cells was also observed, but only at the 48-hour and 96-hour time points. In contrast, in DTH the numbers of CD3+, CD4+, and mRNA+ cells for IL-2 and interferon-gamma were not elevated until 24 hours after challenge and peaked at 48 hours after injection. At 48 hours there was an additional small but significant increase in IL-4 and IL-5 mRNA+ cells. For both LPR and DTH the kinetics of the increases in inflammatory cells and cytokine mRNA-expressing cells paralleled the clinical response. CONCLUSIONS In LPR accumulation of T cells and granulocytes, together with cells expressing mRNA encoding for TH2-type cytokines, is relatively rapid (i.e., within 1 to 6 hours), whereas in DTH the T cell/macrophage infiltration and appearance of cells expressing TH1-type cytokines are not apparent until 24 to 48 hours. In LPR there is a TH1-type (or possibly TH0) component at 48 to 96 hours, and in DTH there is an additional TH2/TH0 response.

Inhibition by Nedocromil Sodium of IgE-Mediated Activation of Human Mononuclear Phagocytes and Platelets in Allergy

The demonstration of a specific receptor for IgE on nonmast cell or basophil leukocytes, such as mononuclear phagocytes, eosinophils, and platelets, suggests that these cells may participate directly in immunological disorders of allergy. Thus, a full understanding of the mode of action of antiallergic or antiasthma drugs must take into account their activity on these nonmast cell leukocytes. Consequently, inhibition by nedocromil sodium of IgE-dependent activation of human alveolar macrophages, blood monocytes and platelets, was investigated. This compound induced an inhibition of the IgE-mediated generation of cytotoxic molecules from monocytes and platelets, together with a concomitant inhibition of their oxidative metabolism, measured by chemiluminescence, and a reduction of the potential ability of alveolar macrophages to synthesize and release mediators, estimated by lysosomal beta-glucuronidase activity. These observations confirm the hypothesis that nedocromil sodium acts on a cell compartment other than the classical mast cell population, in IgE-dependent allergy and, more particularly, in asthma.

Mu opioid receptor expression is increased in inflammatory bowel diseases: implications for homeostatic intestinal inflammation

Background and aims: Recent studies with μ opioid receptor (MOR) deficient mice support a physiological anti-inflammatory effect of MOR at the colon interface. To better understand the potential pharmacological effect of certain opiates in inflammatory bowel diseases (IBD), we (1) evaluated the regulation in vivo and in vitro of human MOR expression by inflammation; and (2) tested the potential anti-inflammatory function of a specific opiate (DALDA) in inflamed and resting human mucosa. Patients and methods: Expression of MOR mRNA and protein was evaluated in healthy and inflamed small bowel and colonic tissues, isolated peripheral blood mononuclear cells and purified monocytes, and CD4+ and CD8+ T cells from healthy donors and IBD patients. The effect of cytokines and nuclear factor κB (NFκB) activation on MOR expression in lymphocyte T and monocytic human cell lines was assessed. Finally, DALDA induced anti-inflammatory effect was investigated in mucosal explants from controls and IBD patients. Results: MOR was expressed in ileal and colonic enteric neurones as well as in immunocytes such as myeloid cells and CD4+ and CD8+ T cells. Overexpressed in active IBD mucosa, MOR was significantly enhanced by cytokines and repressed by NFκB inhibitor in myeloid and lymphocytic cell lines. Furthermore, ex vivo DALDA treatment dampened tumour necrosis factor α mRNA expression in the colon of active IBD patients. Conclusions: Given the increased expression of MOR and the ex vivo beneficial effect of DALDA in active IBD, natural and/or synthetic opioid agonists could help to prevent overt pathological intestinal inflammation.

VENOM IMMUNOTHERAPY MODULATES INTERLEUKIN-4 AND INTERFERON-GAMMA MESSENGER RNA EXPRESSION OF PERIPHERAL T LYMPHOCYTES

The mechanism by which specific immunotherapy exerts its beneficial effect remains unclear. In order to evaluate the influence of venom immunotherapy on the T‐cell cytokine pattern of allergic reactions, we studied interleukin‐4 (IL‐4) and interferon‐γ (IFN‐γ) mRNA expression of peripheral T lymphocytes from 12 patients undergoing rush venom desensitization, before treatment at Day 0 (D0), at Day 15 (D15) and Day 90 (D90) after treatment, and from seven controls. Antigen‐specific T‐cell proliferation was also determined. Cytokine mRNA expression was evaluated using in situ hybridization, 24 hr after culture of peripheral T cells with medium, venom, or an unrelated allergen. Allergen‐induced T‐cell proliferation decreased at D15 and D90 of rush immunotherapy (P ≤ 0.02). In venom‐stimulated cultures of the patient group, there was a decrease in IL‐4 mRNA‐positive cells at D15 and D90 (P ≤ 0.001). Before desensitization, IFN‐γ mRNA expression was lower in patients than in controls and did not increase after in vitro allergen stimulation. In contrast, after immunotherapy, spontaneous IFN‐γ mRNA expression increased, but only at D90 (P ≤ 0.001). The cytokine pattern observed at D90 after immunotherapy was similar to that observed in control subjects. In conclusion, venom immunotherapy induced an altered cytokine mRNA pattern in allergen‐stimulated T cells which was dissociated from the early changes of allergen‐induced T‐cell responsiveness.

Involvement of CCL18 in Allergic Asthma

Allergic asthma is associated with a pulmonary recruitment of Th type 2 cells, basophils, and eosinophils, mainly linked to chemokine production. CCL18 is a chemokine preferentially expressed in the lung, secreted by APCs, induced by Th2-type cytokines, and only present in humans. Therefore, CCL18 may be involved in allergic asthma. PBMC from asthmatics allergic to house dust mite cultured in the presence of Dermatophagoides pteronyssinus 1 (Der p 1) allergen secreted CCL18, 48 and 72 h after stimulation, whereas those from healthy donors did not. Part of CCL18 was directly derived from Der p 1-stimulated plasmacytoid dendritic cells, whereas the other part was linked to monocyte activation by IL-4 and IL-13 produced by Der p 1-stimulated T cells. In bronchoalveolar lavages from untreated asthmatic allergic patients, CCL18 was highly increased compared with controls. Functionally, CCL18 preferentially attracted in vitro-polarized Th2 cells and basophils, but not eosinophils and Th1 cells, and induced basophil histamine and intracellular calcium release. These data show a new function for CCL18, i.e., the recruitment of Th2 cells and basophils, and suggest that CCL18 may play a predominant role in allergic asthma.

Effects of diesel organic extracts on chemokine production by peripheral blood mononuclear cells

BACKGROUND Polyaromatic hydrocarbons (PAHs) associated with diesel exhaust particles (DEPs) are found in the atmospheric urban pollution. Such compounds have been shown to favor IgE production, bronchial hyperresponsiveness, and airway inflammation. Chemokines are a group of chemotactic cytokines involved in the recruitment of inflammatory cells. OBJECTIVE We investigated the effect of DEP-PAHs on the release and mRNA expression of IL-8, MCP-1, and RANTES by PBMCs obtained from healthy subjects. METHODS Protein production in supernatants was assessed by ELISA, and mRNA expression was evaluated by semiquantitative RT-PCR. RESULTS Secretion of IL-8 and RANTES increased in a dose-dependent manner with increasing concentrations of DEP-PAHs (range, 0.5 ng to 50 ng/mL). On the contrary, the release of MCP-1 was significantly inhibited, also in a dose-dependent manner. Messenger RNA production coding for IL-8, RANTES, and MCP-1 showed parallel variations to the production of the correspondent proteins. Effects of DEP-PAHs became significant at 7 hours and up to 48 hours time culture for MCP-1, and up to 24 hours time culture for IL-8 and RANTES. Moreover, supernatants from DEP-PAH-activated cells, compared with those of controls, exhibited a significantly enhanced chemotactic activity for neutrophils and eosinophils, which was significantly inhibited by pretreatment with anti-IL-8 and anti-RANTES neutralizing antibodies, respectively. CONCLUSION These findings suggest that the chemokine pathways are modulated by DEP-PAHs at the transcriptional level, reinforcing the idea that the development of inflammatory reactions might be affected by diesel exhaust emission.