Effect of transcription factor GATA-2 on phagocytic activity of alveolar macrophages from Pneumocystis carinii-infected hosts

Abstract

Alveolar macrophages from Pneumocystis carinii-infected hosts are defective in phagocytosis (W. Chen, J. W. Mills, and A. G. Harmsen, Int. J. Exp. Pathol. 73:709-720, 1992; H. Koziel et al., J. Clin. Investig. 102:1332-1344, 1998). Experiments were performed to determine whether this defect is specific for P. carinii organisms. The results showed that these macrophages were unable to phagocytose both P. carinii organisms and fluorescein isothiocyanate (FITC)-conjugated latex beads, indicating that alveolar macrophages from P. carinii-infected hosts have a general defect in phagocytosis. To determine whether this defect correlates with the recently discovered down-regulation of the GATA-2 transcription factor gene during P. carinii infection, alveolar macrophages from dexamethasone-suppressed or healthy rats were treated with anti-GATA-2 oligonucleotides and then assayed for phagocytosis. Aliquots of the alveolar macrophages were also treated with the sense oligonucleotides as the control. Cells treated with the antisense oligonucleotides were found to have a 46% reduction in phagocytosis of P. carinii organisms and a 65% reduction in phagocytosis of FITC-latex beads compared to those treated with the sense oligonucleotides. To determine whether the defect in phagocytosis in alveolar macrophages from P. carinii-infected hosts can be corrected by overexpression of GATA-2, a plasmid containing the rat GATA-2 gene in the sense orientation driven by the cytomegalovirus (CMV) promoter was introduced into alveolar macrophages from P. carinii-infected rats. Aliquots of the same cells transfected with a plasmid containing GATA-2 in the antisense orientation relative to the CMV promoter served as the control. Alveolar macrophages treated with the sense GATA-2 expression construct were found to increase their phagocytic activity by 66% in phagocytosis of P. carinii organisms and by 280% in phagocytosis of FITC-latex beads compared to those that received the antisense GATA-2 construct. The results of this study indicate that GATA-2 plays an important role in the regulation of phagocytosis in alveolar macrophages during P. carinii infection.

FIG. 1.

Phagocytosis of radiolabeled P. carinii and FITC-labeled latex beads by alveolar macrophages from rats. Alveolar macrophages from normal, Dex-suppressed, and P. carinii-infected rats were isolated and assayed for phagocytic activity with radiolabeled P. carinii trophozoites and FITC-labeled 1-μm-diameter latex beads. Bars represent phagocytosis of radiolabeled P. carinii, and diamonds represent phagocytosis of FITC-labeled latex beads. Numbers in the bars represent the percentage of phagocytically active macrophages from that condition. Results are expressed as the number of P. carinii trophozoites ingested by one million alveolar macrophages or the number of beads ingested per alveolar macrophage and are averages and standard deviations for triplicate reactions from at least three separate experiments.

FIG. 2.

Phagocytosis of radiolabeled P. carinii organisms and FITC-labeled latex beads in alveolar macrophages treated with GATA-2 oligonucleotides. Alveolar macrophages (AMs) from normal and Dex-suppressed rats were treated with sense or antisense GATA-2 oligonucleotides. Bars represent phagocytosis of radiolabeled P. carinii, and diamonds represent phagocytosis of FITC-labeled latex beads. Numbers in the bars represent the percentage of phagocytically active macrophages from that condition. Results are expressed as the number of P. carinii trophozoites ingested by one million alveolar macrophages or the number of beads ingested per alveolar macrophage and are averages and standard deviations for triplicate reactions from at least three separate experiments.

FIG. 3.

Production of GATA-2 protein in rat alveolar macrophages incubated with GATA-2 antisense oligonucleotide. Alveolar macrophages from normal rats were treated with PBS (lane N) or with sense (lane S) or antisense (lane AS) GATA-2 oligonucleotides and then examined for GATA-2 production by Western blotting with an antibody against the rat GATA-2 protein. The same blot was also reacted with an antibody against the GAPDH protein.

FIG. 4.

Nucleotide sequence comparison of the coding regions of the rat and mouse GATA-2 genes. The coding regions of these two genes have exactly the same number of nucleotides. The nucleic acid sequence of the mouse GATA-2 gene is shown in the top lines, and differences in the rat GATA-2 sequence are shown in the bottom lines. Position numbers of nucleotides are shown on the right.

FIG. 5.

Comparison of deduced amino acid sequences of mouse and rat GATA-2. One-letter amino acid codes are used. The amino acid sequence of the mouse GATA-2 gene is shown in the top lines, and differences in the rat GATA-2 sequence are shown in the bottom lines. Amino acid sequences in boldface are the two zinc finger DNA-binding domains of GATA-2 (positions 295 to 319 and 349 to 373). Position numbers of amino acids are shown on the right.

FIG. 6.

Phagocytosis of radiolabeled P. carinii organisms and FITC-labeled latex beads in alveolar macrophages treated with GATA-2 expression vectors. Alveolar macrophages from P. carinii-infected rats were treated with sense or antisense GATA-2 expression vectors. Bars represent phagocytosis of radiolabeled P. carinii, and diamonds represent phagocytosis of FITC-labeled latex beads. Numbers in the bars represent the percentage of phagocytically active macrophages from that condition. Results are expressed as the number of P. carinii trophozoites ingested by one million alveolar macrophages or the number of beads ingested per alveolar macrophage and are averages and standard deviations for triplicate reactions from at least three separate experiments.

FIG. 7.

Expression of GATA-2 in alveolar macrophages from P. carinii-infected rats incubated with GATA-2 expression vector. Alveolar macrophages from Dex-suppressed rats were transfected with the vector pCEP4 (lane C), pGATA2antisense (lane AS), or pGATA2sense (lane S) and then examined for GATA-2 production by Western blotting with an antibody against the rat GATA-2 protein. The same blot was also reacted with an antibody against the GAPDH protein.