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Alveolar macrophage-mediated killing of Pneumocystis carinii f. sp. muris involves molecular recognition by the Dectin-1 beta-glucan receptor.

Steele C, Marrero L, Swain S, Harmsen AG, Zheng M, Brown GD, Gordon S, Shellito JE, Kolls JK - J. Exp. Med. (2003)

Bottom Line: In the presence of Dectin-1 blockage, killing of opsonized P. carinii could be restored through FcgammaRII/III receptors.Opsonized P. carinii could also be efficiently killed in the presence of FcgammaRII/III receptor blockage through Dectin-1-mediated phagocytosis.Taken together, these results show that nonopsonic phagocytosis and subsequent killing of P. carinii by alveolar macrophages is dependent upon recognition by the Dectin-1 beta-glucan receptor.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Division of Pulmonology, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.

ABSTRACT
Innate immune mechanisms against Pneumocystis carinii, a frequent cause of pneumonia in immunocompromised individuals, are not well understood. Using both real time polymerase chain reaction as a measure of organism viability and fluorescent deconvolution microscopy, we show that nonopsonic phagocytosis of P. carinii by alveolar macrophages is mediated by the Dectin-1 beta-glucan receptor and that the subsequent generation of hydrogen peroxide is involved in alveolar macrophage-mediated killing of P. carinii. The macrophage Dectin-1 beta-glucan receptor colocalized with the P. carinii cyst wall. However, blockage of Dectin-1 with high concentrations of anti-Dectin-1 antibody inhibited binding and concomitant killing of P. carinii by alveolar macrophages. Furthermore, RAW 264.7 macrophages overexpressing Dectin-1 bound P. carinii at a higher level than control RAW cells. In the presence of Dectin-1 blockage, killing of opsonized P. carinii could be restored through FcgammaRII/III receptors. Opsonized P. carinii could also be efficiently killed in the presence of FcgammaRII/III receptor blockage through Dectin-1-mediated phagocytosis. We further show that Dectin-1 is required for P. carinii-induced macrophage inflammatory protein 2 production by alveolar macrophages. Taken together, these results show that nonopsonic phagocytosis and subsequent killing of P. carinii by alveolar macrophages is dependent upon recognition by the Dectin-1 beta-glucan receptor.

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P. carinii binding and MIP-2 production by RAW 264.7 macrophages overexpressing Dectin-1. In A, an in vitro binding assay was performed with RAW-FB or RAW-Dectin macrophages prelabeled with an isotype control antibody or 2.0 μg/ml 2A11 and FITC-labeled P. carinii. Slidebook™ analysis software was used to capture, deconvolve, and superimpose FITC-P. carinii images with Nomarski macrophage images. In B, numbers of macrophages bound with P. carinii were enumerated using Slidebook™ software. In C, RAW-FB or RAW-Dectin macrophages were cocultured for 16 h in the presence of rat IgG or 2A11 with P. carinii at a macrophage to total P. carinii organism ratio of 1:100. Controls included macrophages cultured in medium alone. Thereafter, MIP-2 concentrations in coculture supernatants were determined by ELISA. Cumulative results from three separate experiments are shown. *, significant differences between cocultures of rat IgG and 2A11 (P < 0.05). Data are expressed as mean MIP-2 pg/ml ± SEM.
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fig7: P. carinii binding and MIP-2 production by RAW 264.7 macrophages overexpressing Dectin-1. In A, an in vitro binding assay was performed with RAW-FB or RAW-Dectin macrophages prelabeled with an isotype control antibody or 2.0 μg/ml 2A11 and FITC-labeled P. carinii. Slidebook™ analysis software was used to capture, deconvolve, and superimpose FITC-P. carinii images with Nomarski macrophage images. In B, numbers of macrophages bound with P. carinii were enumerated using Slidebook™ software. In C, RAW-FB or RAW-Dectin macrophages were cocultured for 16 h in the presence of rat IgG or 2A11 with P. carinii at a macrophage to total P. carinii organism ratio of 1:100. Controls included macrophages cultured in medium alone. Thereafter, MIP-2 concentrations in coculture supernatants were determined by ELISA. Cumulative results from three separate experiments are shown. *, significant differences between cocultures of rat IgG and 2A11 (P < 0.05). Data are expressed as mean MIP-2 pg/ml ± SEM.

Mentions: RAW-264.7 cells, a murine macrophage cell line, express low endogenous levels of Dectin-1 (26). To confirm by additional means that Dectin-1 was indeed the receptor required for recognition and binding of P. carinii, we used RAW 264.7 macrophages transduced with a retrovirus encoding the murine Dectin-1 cDNA (RAW-Dectin cells; reference 26). Thereafter, we performed an in vitro binding assay using FITC-conjugated P. carinii and quantified the level of P. carinii binding. As shown in the micrographs in Fig. 7 A, there was little to no binding or association of P. carinii cysts with RAW-FB macrophages transduced with a control plasmid (Fig. 7 A, left), whereas RAW-Dectin macrophages overexpressing Dectin-1 were capable of binding P. carinii (Fig. 7 A, middle). Furthermore, when macrophages overexpressing Dectin-1 were pretreated with 2.0 μg/ml 2A11, a significant reduction in binding of P. carinii was observed (Fig. 7 A, right). Quantitative data of the microscopic analysis is presented in Fig. 7 B.


Alveolar macrophage-mediated killing of Pneumocystis carinii f. sp. muris involves molecular recognition by the Dectin-1 beta-glucan receptor.

Steele C, Marrero L, Swain S, Harmsen AG, Zheng M, Brown GD, Gordon S, Shellito JE, Kolls JK - J. Exp. Med. (2003)

P. carinii binding and MIP-2 production by RAW 264.7 macrophages overexpressing Dectin-1. In A, an in vitro binding assay was performed with RAW-FB or RAW-Dectin macrophages prelabeled with an isotype control antibody or 2.0 μg/ml 2A11 and FITC-labeled P. carinii. Slidebook™ analysis software was used to capture, deconvolve, and superimpose FITC-P. carinii images with Nomarski macrophage images. In B, numbers of macrophages bound with P. carinii were enumerated using Slidebook™ software. In C, RAW-FB or RAW-Dectin macrophages were cocultured for 16 h in the presence of rat IgG or 2A11 with P. carinii at a macrophage to total P. carinii organism ratio of 1:100. Controls included macrophages cultured in medium alone. Thereafter, MIP-2 concentrations in coculture supernatants were determined by ELISA. Cumulative results from three separate experiments are shown. *, significant differences between cocultures of rat IgG and 2A11 (P < 0.05). Data are expressed as mean MIP-2 pg/ml ± SEM.
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fig7: P. carinii binding and MIP-2 production by RAW 264.7 macrophages overexpressing Dectin-1. In A, an in vitro binding assay was performed with RAW-FB or RAW-Dectin macrophages prelabeled with an isotype control antibody or 2.0 μg/ml 2A11 and FITC-labeled P. carinii. Slidebook™ analysis software was used to capture, deconvolve, and superimpose FITC-P. carinii images with Nomarski macrophage images. In B, numbers of macrophages bound with P. carinii were enumerated using Slidebook™ software. In C, RAW-FB or RAW-Dectin macrophages were cocultured for 16 h in the presence of rat IgG or 2A11 with P. carinii at a macrophage to total P. carinii organism ratio of 1:100. Controls included macrophages cultured in medium alone. Thereafter, MIP-2 concentrations in coculture supernatants were determined by ELISA. Cumulative results from three separate experiments are shown. *, significant differences between cocultures of rat IgG and 2A11 (P < 0.05). Data are expressed as mean MIP-2 pg/ml ± SEM.
Mentions: RAW-264.7 cells, a murine macrophage cell line, express low endogenous levels of Dectin-1 (26). To confirm by additional means that Dectin-1 was indeed the receptor required for recognition and binding of P. carinii, we used RAW 264.7 macrophages transduced with a retrovirus encoding the murine Dectin-1 cDNA (RAW-Dectin cells; reference 26). Thereafter, we performed an in vitro binding assay using FITC-conjugated P. carinii and quantified the level of P. carinii binding. As shown in the micrographs in Fig. 7 A, there was little to no binding or association of P. carinii cysts with RAW-FB macrophages transduced with a control plasmid (Fig. 7 A, left), whereas RAW-Dectin macrophages overexpressing Dectin-1 were capable of binding P. carinii (Fig. 7 A, middle). Furthermore, when macrophages overexpressing Dectin-1 were pretreated with 2.0 μg/ml 2A11, a significant reduction in binding of P. carinii was observed (Fig. 7 A, right). Quantitative data of the microscopic analysis is presented in Fig. 7 B.

Bottom Line: In the presence of Dectin-1 blockage, killing of opsonized P. carinii could be restored through FcgammaRII/III receptors.Opsonized P. carinii could also be efficiently killed in the presence of FcgammaRII/III receptor blockage through Dectin-1-mediated phagocytosis.Taken together, these results show that nonopsonic phagocytosis and subsequent killing of P. carinii by alveolar macrophages is dependent upon recognition by the Dectin-1 beta-glucan receptor.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Division of Pulmonology, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.

ABSTRACT
Innate immune mechanisms against Pneumocystis carinii, a frequent cause of pneumonia in immunocompromised individuals, are not well understood. Using both real time polymerase chain reaction as a measure of organism viability and fluorescent deconvolution microscopy, we show that nonopsonic phagocytosis of P. carinii by alveolar macrophages is mediated by the Dectin-1 beta-glucan receptor and that the subsequent generation of hydrogen peroxide is involved in alveolar macrophage-mediated killing of P. carinii. The macrophage Dectin-1 beta-glucan receptor colocalized with the P. carinii cyst wall. However, blockage of Dectin-1 with high concentrations of anti-Dectin-1 antibody inhibited binding and concomitant killing of P. carinii by alveolar macrophages. Furthermore, RAW 264.7 macrophages overexpressing Dectin-1 bound P. carinii at a higher level than control RAW cells. In the presence of Dectin-1 blockage, killing of opsonized P. carinii could be restored through FcgammaRII/III receptors. Opsonized P. carinii could also be efficiently killed in the presence of FcgammaRII/III receptor blockage through Dectin-1-mediated phagocytosis. We further show that Dectin-1 is required for P. carinii-induced macrophage inflammatory protein 2 production by alveolar macrophages. Taken together, these results show that nonopsonic phagocytosis and subsequent killing of P. carinii by alveolar macrophages is dependent upon recognition by the Dectin-1 beta-glucan receptor.

Show MeSH
Related in: MedlinePlus