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Defective Expression of Fas Messenger RNA and Fas Receptor on Pulmonary T Cells from Patients with Asthma

Fabrizio Spinozzi, MD; Marco Fizzotti, MD; Elisabetta Agea, MD; Simonetta Piattoni, BS; Sara Droetto, BS; Anna Russano, BS; Nicolino Forenza, MD; Gabrio Bassotti, MD, PhD; Fausto Grignani, MD; and Alberto Bertotto, MD
[+] Article and Author Information

From University of Perugia, Perugia, Italy. Grant Support: In part by a grant from the Italian Ministry of University, Scientific and Technologic Research (Dr. Spinozzi). Requests for Reprints: Fabrizio Spinozzi, MD, Dipartimento di Medicina Clinica e Sperimentale, Sezione di Medicina Interna e Scienze Oncologiche, Policlinico Monteluce, I-06122 Perugia, Italy. Current Author Addresses: Drs. Spinozzi, Fizzotti, Agea, Piattoni, Droetto, Russano, and Grignani: Dipartimento di Medicina Clinica e Sperimentale, Sezione di Medicina Interna e Scienze Oncologiche, Universita di Perugia, Policlinico Monteluce, I-06122 Perugia, Italy.


Copyright ©2004 by the American College of Physicians


Ann Intern Med. 1998;128(5):363-369. doi:10.7326/0003-4819-128-5-199803010-00004
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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.

Figures

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Figure 1.
In vitro induction of programmed cell death by IgM anti-Fas monoclonal antibody. Top.PBottom.

Quantitative assay with propidium iodide staining and flow cytometry. Enriched bronchoalveolar lavage T cells from patients with asthma and from controls were incubated for 24 hours with IgM anti-Fas monoclonal antibody, 0.5 µg/mL (Immunotech, Marseille, France), and then stained with hypotonic propidium iodide solution. Culturing in medium alone for 24 hours did not induce apoptosis in significant percentages of cells, but in vitro stimulation with anti-Fas monoclonal antibody determined the appearance of significant percentages of cells with hypodiploid DNA in samples from controls. Black bars represent patients with asthma; white bars represent controls. *  < 0.001 for comparison with patients with asthma. A representative qualitative assay of DNA fragmentation of T-cell samples from patients with asthma and controls loaded into wells of 1% agarose gel. Ethidium bromide staining was used to visualize DNA. Lane 1: control, medium alone; lane 2: patient with asthma, anti-Fas monoclonal antibody; lane 3: control, anti-Fas monoclonal antibody; lane 4: patient with asthma, anti-Fas monoclonal antibody; lane 5: control, anti-Fas monoclonal antibody. A clear DNA ladder, characteristic of apoptosis, is seen for anti-Fas-treated T cells from controls but not patients with asthma. Numbers in first column are the molecular weight.

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Figure 2.
Reverse transcription polymerase chain reaction. Top.Bottom.

Fas messenger RNA (mRNA) isolated from enriched bronchoalveolar lavage T cells. Lane 1: control; lanes 2, 3, and 4: patients with asthma; lane 5: control; lane 6: U937 cell line as a positive control. Samples from patients with asthma, unlike those from controls, lack Fas mRNA. β-actin mRNA from the same samples. Numbers in first column are the molecular weight.

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Figure 3.
Modulation of surface Fas expression by cytokines.topbottom

Representative FACscan profiles (Becton-Dickinson, Mountain View, California) obtained in a patient with asthma ( ) and a control ( ). Pulmonary enriched T cells were cultured for 24 hours with plastic-coated anti-CD3 monoclonal antibody plus recombinant human interleukin-2, 50 U/mL (top left and bottom left), or recombinant human interleukin-4, 400 U/mL (top right and bottom right). Subsequent surface Fas expression was measured on cytofluorometry by gating positively stained cells (M1) in the FL2 channel of the FACScan. Relative percentages and fluorescence intensity are shown. In vitro anti-CD3 stimulation was able to upregulate Fas molecule on enriched T cells. However, although interleukin-2 maintained upregulation of the molecule, interleukin-4 determined its partial downregulation. This was seen in patients with asthma but was almost absent from controls. One experiment representative of all those performed on both patients and controls is shown.

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