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Specific migratory dendritic cells rapidly transport antigen from the airways to the thoracic lymph nodes.

Vermaelen KY, Carro-Muino I, Lambrecht BN, Pauwels RA - J. Exp. Med. (2001)

Bottom Line: After instillation, FITC(+) cells with stellate morphology were found deep in the TLN T cell area.AW-LNDCs were depleted by 7 d of ganciclovir treatment in thymidine kinase transgenic mice, resulting in a strong reduction of FITC-macromolecule transport into the TLNs.These results validate the unique sentinel role of airway DCs, picking up antigen in the airways and delivering it in an immunogenic form to the T cells in the TLNs.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory Diseases, Ghent University Hospital, Ghent B-9000, Belgium. karim.vermaelen@rug.ac.be

ABSTRACT
Antigen transport from the airway mucosa to the thoracic lymph nodes (TLNs) was studied in vivo by intratracheal instillation of fluorescein isothiocyanate (FITC)-conjugated macromolecules. After instillation, FITC(+) cells with stellate morphology were found deep in the TLN T cell area. Using flow cytometry, an FITC signal was exclusively detected in CD11c(med-hi)/major histocompatibility complex class II (MHCII)(hi) cells, representing migratory airway-derived lymph node dendritic cells (AW-LNDCs). No FITC signal accumulated in lymphocytes and in a CD11c(hi)MHCII(med) DC group containing a CD8 alpha(hi) subset (non-airway-derived [NAW]-LNDCs). Sorted AW-LNDCs showed long MHCII(bright) cytoplasmic processes and intracytoplasmatic FITC(+) granules. The fraction of FITC(+) AW-LNDCs peaked after 24 h and had reached baseline by day 7. AW-LNDCs were depleted by 7 d of ganciclovir treatment in thymidine kinase transgenic mice, resulting in a strong reduction of FITC-macromolecule transport into the TLNs. Compared with intrapulmonary DCs, AW-LNDCs had a mature phenotype and upregulated levels of MHCII, B7-2, CD40, and intracellular adhesion molecule (ICAM)-1. In addition, sorted AW-LNDCs from FITC-ovalbumin (OVA)-instilled animals strongly presented OVA to OVA-TCR transgenic T cells. These results validate the unique sentinel role of airway DCs, picking up antigen in the airways and delivering it in an immunogenic form to the T cells in the TLNs.

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(A) Identification of DCs in collagenase digests of whole lungs (parenchymal tissue and large airways including trachea, no bronchoalveolar lavage) after collagenase/DNase/EDTA treatment. Pulmonary DCs, defined as CD11c+/low autofluorescence cells, were found to be MHCII+, in contrast to CD11c+/high autofluorescence cells. (B) CD11c+ TLN cells obtained using the same organ digestion protocol show even higher MHCII expression.
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Figure 8: (A) Identification of DCs in collagenase digests of whole lungs (parenchymal tissue and large airways including trachea, no bronchoalveolar lavage) after collagenase/DNase/EDTA treatment. Pulmonary DCs, defined as CD11c+/low autofluorescence cells, were found to be MHCII+, in contrast to CD11c+/high autofluorescence cells. (B) CD11c+ TLN cells obtained using the same organ digestion protocol show even higher MHCII expression.

Mentions: We compared the phenotype of lung and TLN DCs after collagenase digestion and EDTA treatment of whole lungs and corresponding TLNs. We chose to phenotype cells right after organ digestion in order to avoid artefacts originating from additional DC enrichment and culture procedures. AW and NAW DC subsets in the LNs were outlined using the MHCII/CD11c double staining as described above. To pinpoint DCs in the lungs, we used a strategy based on findings by Havenith et al. 35 in which DCs and monocytic DC precursors were identified within the low autofluorescent cell fraction of rat bronchoalveolar lavage fluid. Additionally, in this study the phenotype of the low autofluorescent cell fraction was found to shift further towards typical mature DC morphology after overnight incubation. We applied this approach to murine lung cells and added the expression of CD11c to the immunofluorescent identification criteria (Fig. 8 A). Two peaks of autofluorescence could be distinguished within the CD11c+ gate. When sorted, CD11c+/low autofluorescence cells showed a predominant monocyte and immature DC morphology. After overnight incubation in cytokine-free, serum-supplemented medium this appearance shifted clearly towards typical DCs with numerous cytoplasmic extensions (data not shown). We did not consider high levels of surface MHCII as a DC identification criterion a priori in order to avoid a bias towards more mature forms. Nevertheless, CD11c+/low autofluorescence cells were >90% MHCII+. Therefore, these cells are hereafter referred to as “intrapulmonary DCs.” The mean fluorescence intensity of MHCII was even higher for CD11c+ TLN cells (Fig. 8 B).


Specific migratory dendritic cells rapidly transport antigen from the airways to the thoracic lymph nodes.

Vermaelen KY, Carro-Muino I, Lambrecht BN, Pauwels RA - J. Exp. Med. (2001)

(A) Identification of DCs in collagenase digests of whole lungs (parenchymal tissue and large airways including trachea, no bronchoalveolar lavage) after collagenase/DNase/EDTA treatment. Pulmonary DCs, defined as CD11c+/low autofluorescence cells, were found to be MHCII+, in contrast to CD11c+/high autofluorescence cells. (B) CD11c+ TLN cells obtained using the same organ digestion protocol show even higher MHCII expression.
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Related In: Results  -  Collection

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Figure 8: (A) Identification of DCs in collagenase digests of whole lungs (parenchymal tissue and large airways including trachea, no bronchoalveolar lavage) after collagenase/DNase/EDTA treatment. Pulmonary DCs, defined as CD11c+/low autofluorescence cells, were found to be MHCII+, in contrast to CD11c+/high autofluorescence cells. (B) CD11c+ TLN cells obtained using the same organ digestion protocol show even higher MHCII expression.
Mentions: We compared the phenotype of lung and TLN DCs after collagenase digestion and EDTA treatment of whole lungs and corresponding TLNs. We chose to phenotype cells right after organ digestion in order to avoid artefacts originating from additional DC enrichment and culture procedures. AW and NAW DC subsets in the LNs were outlined using the MHCII/CD11c double staining as described above. To pinpoint DCs in the lungs, we used a strategy based on findings by Havenith et al. 35 in which DCs and monocytic DC precursors were identified within the low autofluorescent cell fraction of rat bronchoalveolar lavage fluid. Additionally, in this study the phenotype of the low autofluorescent cell fraction was found to shift further towards typical mature DC morphology after overnight incubation. We applied this approach to murine lung cells and added the expression of CD11c to the immunofluorescent identification criteria (Fig. 8 A). Two peaks of autofluorescence could be distinguished within the CD11c+ gate. When sorted, CD11c+/low autofluorescence cells showed a predominant monocyte and immature DC morphology. After overnight incubation in cytokine-free, serum-supplemented medium this appearance shifted clearly towards typical DCs with numerous cytoplasmic extensions (data not shown). We did not consider high levels of surface MHCII as a DC identification criterion a priori in order to avoid a bias towards more mature forms. Nevertheless, CD11c+/low autofluorescence cells were >90% MHCII+. Therefore, these cells are hereafter referred to as “intrapulmonary DCs.” The mean fluorescence intensity of MHCII was even higher for CD11c+ TLN cells (Fig. 8 B).

Bottom Line: After instillation, FITC(+) cells with stellate morphology were found deep in the TLN T cell area.AW-LNDCs were depleted by 7 d of ganciclovir treatment in thymidine kinase transgenic mice, resulting in a strong reduction of FITC-macromolecule transport into the TLNs.These results validate the unique sentinel role of airway DCs, picking up antigen in the airways and delivering it in an immunogenic form to the T cells in the TLNs.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory Diseases, Ghent University Hospital, Ghent B-9000, Belgium. karim.vermaelen@rug.ac.be

ABSTRACT
Antigen transport from the airway mucosa to the thoracic lymph nodes (TLNs) was studied in vivo by intratracheal instillation of fluorescein isothiocyanate (FITC)-conjugated macromolecules. After instillation, FITC(+) cells with stellate morphology were found deep in the TLN T cell area. Using flow cytometry, an FITC signal was exclusively detected in CD11c(med-hi)/major histocompatibility complex class II (MHCII)(hi) cells, representing migratory airway-derived lymph node dendritic cells (AW-LNDCs). No FITC signal accumulated in lymphocytes and in a CD11c(hi)MHCII(med) DC group containing a CD8 alpha(hi) subset (non-airway-derived [NAW]-LNDCs). Sorted AW-LNDCs showed long MHCII(bright) cytoplasmic processes and intracytoplasmatic FITC(+) granules. The fraction of FITC(+) AW-LNDCs peaked after 24 h and had reached baseline by day 7. AW-LNDCs were depleted by 7 d of ganciclovir treatment in thymidine kinase transgenic mice, resulting in a strong reduction of FITC-macromolecule transport into the TLNs. Compared with intrapulmonary DCs, AW-LNDCs had a mature phenotype and upregulated levels of MHCII, B7-2, CD40, and intracellular adhesion molecule (ICAM)-1. In addition, sorted AW-LNDCs from FITC-ovalbumin (OVA)-instilled animals strongly presented OVA to OVA-TCR transgenic T cells. These results validate the unique sentinel role of airway DCs, picking up antigen in the airways and delivering it in an immunogenic form to the T cells in the TLNs.

Show MeSH
Related in: MedlinePlus