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First published online July 2, 2007
doi: 10.1242/10.1242/jcs.000331

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Expression of the transcription factor NFAT2 in human neutrophils: IgE-dependent, Ca²⁺- and calcineurin-mediated NFAT2 activation

¹ Servicio Regional de Inmunología y Alergia, Hospital Universitario Virgen Macarena, Sevilla, Spain
² Departamento de Bioquímica Médica y Biología Molecular, Facultad de Medicina, Universidad de Sevilla, Sevilla, Spain
³ Fundación IMABIS, Laboratorio de Investigación, Hospital Civil, Málaga, Spain
⁴ Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante, Alicante, Spain

View larger version (26K): [in this window] [in a new window]   Fig. 1. Analysis of NFAT mRNA expression in human neutrophils. Total RNA was extracted from highly purified unstimulated neutrophils from both human healthy donors (N1-N3) and human allergic patients (N4-N6), as well as from PBLs (L) and Jurkat T cells (J) stimulated with 100 nM PMA plus 500 nM ionomycin, and from highly purified eosinophils (E) used as positive and negative controls. (A,B) RNA samples were analysed by conventional RT-PCR, using specific primers for the amplification of the indicated NFAT isoform transcripts (A), or of the Charcot-Leyden crystal protein (C-L) transcripts (B). Results from amplification of the 2-microglobulin (2m) housekeeping gene transcript are shown in both panels. (C) RNA was isolated from neutrophils from allergic patients (AP) or healthy donors (HD) or from PBLs (L). The levels of NFAT1, NFAT2, NFAT4 and NFAT5 mRNAs were analysed by real-time PCR analysis as indicated in the Materials and Methods section. The levels of mRNAs were standardised against the level of 2-microglobulin (2m) mRNA. Values are mean ± s.e.m. from eight individuals in each group.

View larger version (67K): [in this window] [in a new window]   Fig. 2. Expression of the NFAT2 protein isoform in human neutrophils. (A) The potential presence of the five NFAT isoforms was analysed by western blotting of whole-cell extracts from unstimulated neutrophils obtained immediately after their isolation from both human healthy donors (N1-N3) and human allergic patients (N4-N6), as well as from PBLs (L) and Jurkat T cells (J) stimulated with 100 nM plus 500 nM ionomycin used as positive controls. -actin was used as an internal control to verify that equal amounts of protein were loaded per lane. All blots shown are representative of at least three independent experiments in each group of individuals, with similar results. (B) Flow cytometry analysis of NFAT2 expression in neutrophils isolated from an allergic patient (left panel) and a healthy donor (right panel). After isolation, two-colour flow cytometry analysis of CD66b and NFAT2 expression was performed. Results obtained are expressed as the percentage of double-positive (CD66b+ and NFAT2+) cells. Results shown are representative of at least three independent experiments in each different group of individuals.

View larger version (41K): [in this window] [in a new window]   Fig. 3. Anti-IgE-elicited NFAT2 nuclear translocation in human neutrophils. Constitutive NFAT2 expression in human neutrophils. (A) Neutrophils isolated from an allergic patient were stimulated with 10 µg/ml anti-IgE (-IgE) for the indicated times (up to 6 hours), or incubated with 10 µg/ml non-specific goat IgG (IgG) for 4 hours. Then, nuclear and cytosolic extracts were obtained. (B) Whole-cell extracts were obtained immediately after neutrophil isolation from an allergic patient (basal), or neutrophils were cultured in the absence or presence of 10 µg/ml anti-IgE (-IgE) for the indicated times (up to 24 hours), and then whole-cell extracts were obtained. Cellular, cytosolic and nuclear NFAT2 levels were analysed by western blotting, using anti-human NFAT2-specific Abs. -actin was used as an internal control. The blot shown is representative of three independent experiments with similar results. Histograms above each lane show the mean (± s.e.m.) values quantified from the blots from three separate experiments.

View larger version (23K): [in this window] [in a new window]   Fig. 4. Anti-FcRs- and antigen-elicited NFAT2 nuclear translocation in human neutrophils. Neutrophils from three allergic patients sensitised to either T9 (A), E1 (B) or W6 (C) were stimulated for 4 hours with 10 µg/ml anti-IgE (-IgE) or with the indicated specific antigens (W6, D1, D2, G3, T9, E1 or E2) at 10 µg/ml. (D) Neutrophils from an allergic patient were incubated with 5 µg/ml anti-FcRII (-FcRII), anti-Mac-2 (-Mac-2), or anti-FcRI (-FcRI) Abs or mouse IgG1 for 4 hours at 37°C. Then, nuclear extracts were obtained, and NFAT2 levels were analysed by western blotting, using anti-human NFAT2-specific Abs. Equal amounts of protein were loaded per lane. Histograms above each lane show the mean (± s.e.m.) values quantified from the blots from three separate experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 5. Antigens, anti-FcRs and anti-IgE increase cytosolic Ca2+ levels and CaN activity in human neutrophils. (A) Fura-2-AM-loaded neutrophils from an allergic patient sensitised to G3 were treated at the times indicated with 10 µg/ml anti-IgE (-IgE) (1), 10 µg/ml G3 (2), 50 nM fMLP (3), 5 µg/ml anti-galectin-3 (-galectin-3) (4), 5 µg/ml anti-FcRI (-FcRI) (5), or 5 µg/ml anti-FcRII (-FcRII) (6), and cytosolic Ca2+ was spectrofluorometrically recorded for 2 minutes. Each graph is representative of a set of three experiments that yielded similar results. (B) Neutrophils from an allergic patient sensitised to E1 were incubated with 10 µg/ml anti-IgE (-IgE), 10 µg/ml E1 antigen, or unspecific goat IgG (IgG) for the indicated times. Then, the cells were lysed, and CaN activity was assayed. Values are the means ± s.e.m. from four separate experiments performed in triplicate.

View larger version (31K): [in this window] [in a new window]   Fig. 6. Involvement of CaN in IgE-dependent NFAT2 nuclear translocation in human neutrophils. Neutrophils from an allergic patient were pretreated with CsA (A) or VIVIT peptide (B) at the indicated doses for 1 hour, and then treated with 10 µg/ml anti-IgE (-IgE) for 4 hours. Nuclear extracts were obtained, and NFAT2 levels analysed by western blotting, using anti-human NFAT2-specific Abs. Equal amounts of protein were loaded per lane. Histograms above each lane show the mean (± s.e.m.) values quantified from the blots from three separate experiments. (C) Whole-cell lysates (1 mg of protein) from untreated neutrophils from an allergic patient were incubated at 4°C for 18 hours with anti-CaN Abs. After immunoprecipitation (IP), the pellets were analysed by western blotting, using anti-NFAT2 (left upper panel). Thereafter, the blot was stripped, and reprobed with anti-CaN (left lower panel). Immunoprecipitation was carried out as indicated in A, except that anti-NFAT2 Abs was used, and subsequent western blotting analysis was carried out using anti-CaN Abs (right upper panel). After stripping, the blot was reprobed with anti-NFAT2 Abs (right lower panel). The signals directly obtained from neutrophil whole-cell lysates (80 µg) subjected to western blotting analysis without immunoprecipitation (WL) are also shown in both panels.

View larger version (42K): [in this window] [in a new window]   Fig. 7. Anti-IgE-elicited NFAT2 DNA-binding activity in human neutrophils. Role of CaN. (A,B) Neutrophils from an allergic patient were treated either with 10 µg/ml anti-IgE (-IgE) for the indicated times (A), or with the indicated doses of anti-IgE for 4 hours (B). (C) Neutrophils from an allergic patient sensitised to G3 were pretreated with 1 µg/ml CsA or 50 µg/ml VIVIT peptide for 1 hour, and then treated with 10 µg/ml anti-IgE (-IgE) or 10 µg/ml G3 antigen for 4 hours. Nuclear extracts were then obtained, and binding to the 32P-labelled NFAT oligonucleotide probe was assessed by electrophoretic mobility shift assays. Incubation of neutrophils with 10 µg/ml non-specific IgG for 4 hours was also carried out as a negative control (IgG). Assays performed in the presence of 1 µl of anti-NFAT2 (Ab) or carrying a 100-fold molar excess of unlabelled oligonucleotide (*) are also shown.

View larger version (35K): [in this window] [in a new window]   Fig. 8. Involvement of NFAT in IgE-dependent COX2 expression in human neutrophils. Neutrophils from an allergic patient sensitised to G3 antigen were pretreated with CsA or cell-permeable VIVIT, at the indicated doses for 1 hour, and then incubated for 24 hours with 1 µg/ml LPS or 10 µg/ml anti-IgE Ab (-IgE; A) or 10 µg/ml G3 (B). COX2 mRNA levels were analysed by conventional RT-PCR, using COX2- and GAPDH-specific primers; COX2 and -actin protein expression was analysed by western blotting, and the levels of PGE2 were measured in the culture medium supernatants, using an enzyme immunoassay. The values shown are the mean ± s.e.m. from three independent assays in which each measurement was performed in triplicate. All the experiments were performed at least in triplicate.