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Accumulation of immature Langerhans cells in human lymph nodes draining chronically inflamed skin.

Geissmann F, Dieu-Nosjean MC, Dezutter C, Valladeau J, Kayal S, Leborgne M, Brousse N, Saeland S, Davoust J - J. Exp. Med. (2002)

Bottom Line: This indicates that LC migration and maturation can be independently regulated events.We suggest that during DL, inflammatory stimuli in the skin increase the migration of LCs to the lymph node but without associated maturation.Immature LCs might regulate immune responses during chronic inflammation.

View Article: PubMed Central - PubMed

Affiliation: Service d'Anatomie Pathologique, IFR Necker-Enfants Malades, 75015 Paris, France. geissman@necker.fr

ABSTRACT
The coordinated migration and maturation of dendritic cells (DCs) such as intraepithelial Langerhans cells (LCs) is considered critical for T cell priming in response to inflammation in the periphery. However, little is known about the role of inflammatory mediators for LC maturation and recruitment to lymph nodes in vivo. Here we show in human dermatopathic lymphadenitis (DL), which features an expanded population of LCs in one draining lymph node associated with inflammatory lesions in its tributary skin area, that the Langerin/CD207(+) LCs constitute a predominant population of immature DCs, which express CD1a, and CD68, but not CD83, CD86, and DC-lysosomal-associated membrane protein (LAMP)/CD208. Using LC-type cells generated in vitro in the presence of transforming growth factor (TGF)-beta1, we further found that tumor necrosis factor (TNF)-alpha, as a prototype proinflammatory factor, and a variety of inflammatory stimuli and bacterial products, increase Langerin expression and Langerin dependent Birbeck granules formation in cell which nevertheless lack costimulatory molecules, DC-LAMP/CD208 and potent T cell stimulatory activity but express CCR7 and respond to the lymph node homing chemokines CCL19 and CCL21. This indicates that LC migration and maturation can be independently regulated events. We suggest that during DL, inflammatory stimuli in the skin increase the migration of LCs to the lymph node but without associated maturation. Immature LCs might regulate immune responses during chronic inflammation.

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In vitro differentiation of LC-type cells. (A) TNF-α upregulates membrane Langerin expression but not MHC class I, class II, and CD86. Cells were cultured for 5–6 d in the presence of the indicated cyto-kines as indicated in Materials and Methods. 10 ng/ml IL-4 was added at day 0 only. Cells were analyzed by flow cytometry for membrane expression of HLA-ABC, HLA-DR, Langerin, and CD1a and CD86. Dot plots are gated on viable cells. (B) CD40L but not TNF-α induces internalization of Langerin, upregulation of membrane HLA-DR and acquisition of a Mature dendritic shape. Cells cultured as in A are permeabilized and analyzed by confocal microscopy for expression of Langerin (top panel) and DR (bottom panel). Left panels represent cells cultured for 5–6 d in GM-CSF, IL-4 (added at day 0 only), and TGF-β1, middle panels represent cells cultured for 5–6 d in GM-CSF, IL-4 (added at day 0 only), TGF-β1, and TNF-α (10 ng/ml for the last 40 h of culture). The right panels represent cells cultured for 5–6 d in GM-CSF, IL-4 (added at day 0 only), TGF-β1, with CD40L transfected fibroblasts (for the last 40h of culture). Incubation with control fibroblasts (LcCD32) as described in Materials and Methods does not results in the activation of cells (unpublished data). Original magnification: × 400. (C) TNF-α treated LC-type cells express CD68 while CD40L treated LC-type cells express DC-LAMP. Cells cultured and processed as in B were analyzed for expression of Langerin, DC-LAMP and CD68 by three-color confocal microscopy. Left panels represent cells cultured for 5–6 d in GM-CSF, IL-4 (added at day 0 only), TGF-β1 and TNF-α (10 ng/ml for the last 40 h of culture). Right panels represent cells cultured for 5–6 d in GM-CSF, IL-4 (added at day 0 only), TGF-β1, with CD40L transfected fibroblasts (for the last 40 h of culture). Incubation with control fibroblasts (LcCD32) as described in Materials and Methods does not result in the activation of DCs (unpublished data and reference 26).
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fig3: In vitro differentiation of LC-type cells. (A) TNF-α upregulates membrane Langerin expression but not MHC class I, class II, and CD86. Cells were cultured for 5–6 d in the presence of the indicated cyto-kines as indicated in Materials and Methods. 10 ng/ml IL-4 was added at day 0 only. Cells were analyzed by flow cytometry for membrane expression of HLA-ABC, HLA-DR, Langerin, and CD1a and CD86. Dot plots are gated on viable cells. (B) CD40L but not TNF-α induces internalization of Langerin, upregulation of membrane HLA-DR and acquisition of a Mature dendritic shape. Cells cultured as in A are permeabilized and analyzed by confocal microscopy for expression of Langerin (top panel) and DR (bottom panel). Left panels represent cells cultured for 5–6 d in GM-CSF, IL-4 (added at day 0 only), and TGF-β1, middle panels represent cells cultured for 5–6 d in GM-CSF, IL-4 (added at day 0 only), TGF-β1, and TNF-α (10 ng/ml for the last 40 h of culture). The right panels represent cells cultured for 5–6 d in GM-CSF, IL-4 (added at day 0 only), TGF-β1, with CD40L transfected fibroblasts (for the last 40h of culture). Incubation with control fibroblasts (LcCD32) as described in Materials and Methods does not results in the activation of cells (unpublished data). Original magnification: × 400. (C) TNF-α treated LC-type cells express CD68 while CD40L treated LC-type cells express DC-LAMP. Cells cultured and processed as in B were analyzed for expression of Langerin, DC-LAMP and CD68 by three-color confocal microscopy. Left panels represent cells cultured for 5–6 d in GM-CSF, IL-4 (added at day 0 only), TGF-β1 and TNF-α (10 ng/ml for the last 40 h of culture). Right panels represent cells cultured for 5–6 d in GM-CSF, IL-4 (added at day 0 only), TGF-β1, with CD40L transfected fibroblasts (for the last 40 h of culture). Incubation with control fibroblasts (LcCD32) as described in Materials and Methods does not result in the activation of DCs (unpublished data and reference 26).

Mentions: To explore the effect of inflammatory cytokines on LC differentiation and migration, we cultured LC-type cells in the presence of TNF-α. LCs may be derived from CD34+ hemopoietic progenitor-cells (45–48). However, these cells represent a wide range of maturation stages. To synchronize the maturation stages of LCs in the cultures we used mainly LC-type cells derived from human monocytes, grown in the presence of GM-CSF, IL-4, and TGF-β1 (21, 41). This further allowed us to compare LCs with DCi derived from the same monocytes differentiated in the presence of GM-CSF and IL-4 (7, 21, 49). When cultured in the absence of TNF-α approximately one-third of the monocyte-derived LC-type cells expressed significant surface levels of Langerin (Fig. 3 A). Remarkably, the addition of TNF-α at days 5 or 6 of the culture strongly increased the proportion of Langerin+ cells (Fig. 3 A). Langerin+ cells increased from 33 ± 12% to 75 ± 15% of total cells (n = 6 independent experiments), with a considerable higher MFI (from 66 ± 20 to 250 ± 130). However, the presence of TGF-β1 was mandatory for the induction of Langerin and BGs by TNF-α (unpublished data) demonstrating that TNF-α acts in synergy with TGF-β1 to promote LC differentiation.


Accumulation of immature Langerhans cells in human lymph nodes draining chronically inflamed skin.

Geissmann F, Dieu-Nosjean MC, Dezutter C, Valladeau J, Kayal S, Leborgne M, Brousse N, Saeland S, Davoust J - J. Exp. Med. (2002)

In vitro differentiation of LC-type cells. (A) TNF-α upregulates membrane Langerin expression but not MHC class I, class II, and CD86. Cells were cultured for 5–6 d in the presence of the indicated cyto-kines as indicated in Materials and Methods. 10 ng/ml IL-4 was added at day 0 only. Cells were analyzed by flow cytometry for membrane expression of HLA-ABC, HLA-DR, Langerin, and CD1a and CD86. Dot plots are gated on viable cells. (B) CD40L but not TNF-α induces internalization of Langerin, upregulation of membrane HLA-DR and acquisition of a Mature dendritic shape. Cells cultured as in A are permeabilized and analyzed by confocal microscopy for expression of Langerin (top panel) and DR (bottom panel). Left panels represent cells cultured for 5–6 d in GM-CSF, IL-4 (added at day 0 only), and TGF-β1, middle panels represent cells cultured for 5–6 d in GM-CSF, IL-4 (added at day 0 only), TGF-β1, and TNF-α (10 ng/ml for the last 40 h of culture). The right panels represent cells cultured for 5–6 d in GM-CSF, IL-4 (added at day 0 only), TGF-β1, with CD40L transfected fibroblasts (for the last 40h of culture). Incubation with control fibroblasts (LcCD32) as described in Materials and Methods does not results in the activation of cells (unpublished data). Original magnification: × 400. (C) TNF-α treated LC-type cells express CD68 while CD40L treated LC-type cells express DC-LAMP. Cells cultured and processed as in B were analyzed for expression of Langerin, DC-LAMP and CD68 by three-color confocal microscopy. Left panels represent cells cultured for 5–6 d in GM-CSF, IL-4 (added at day 0 only), TGF-β1 and TNF-α (10 ng/ml for the last 40 h of culture). Right panels represent cells cultured for 5–6 d in GM-CSF, IL-4 (added at day 0 only), TGF-β1, with CD40L transfected fibroblasts (for the last 40 h of culture). Incubation with control fibroblasts (LcCD32) as described in Materials and Methods does not result in the activation of DCs (unpublished data and reference 26).
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fig3: In vitro differentiation of LC-type cells. (A) TNF-α upregulates membrane Langerin expression but not MHC class I, class II, and CD86. Cells were cultured for 5–6 d in the presence of the indicated cyto-kines as indicated in Materials and Methods. 10 ng/ml IL-4 was added at day 0 only. Cells were analyzed by flow cytometry for membrane expression of HLA-ABC, HLA-DR, Langerin, and CD1a and CD86. Dot plots are gated on viable cells. (B) CD40L but not TNF-α induces internalization of Langerin, upregulation of membrane HLA-DR and acquisition of a Mature dendritic shape. Cells cultured as in A are permeabilized and analyzed by confocal microscopy for expression of Langerin (top panel) and DR (bottom panel). Left panels represent cells cultured for 5–6 d in GM-CSF, IL-4 (added at day 0 only), and TGF-β1, middle panels represent cells cultured for 5–6 d in GM-CSF, IL-4 (added at day 0 only), TGF-β1, and TNF-α (10 ng/ml for the last 40 h of culture). The right panels represent cells cultured for 5–6 d in GM-CSF, IL-4 (added at day 0 only), TGF-β1, with CD40L transfected fibroblasts (for the last 40h of culture). Incubation with control fibroblasts (LcCD32) as described in Materials and Methods does not results in the activation of cells (unpublished data). Original magnification: × 400. (C) TNF-α treated LC-type cells express CD68 while CD40L treated LC-type cells express DC-LAMP. Cells cultured and processed as in B were analyzed for expression of Langerin, DC-LAMP and CD68 by three-color confocal microscopy. Left panels represent cells cultured for 5–6 d in GM-CSF, IL-4 (added at day 0 only), TGF-β1 and TNF-α (10 ng/ml for the last 40 h of culture). Right panels represent cells cultured for 5–6 d in GM-CSF, IL-4 (added at day 0 only), TGF-β1, with CD40L transfected fibroblasts (for the last 40 h of culture). Incubation with control fibroblasts (LcCD32) as described in Materials and Methods does not result in the activation of DCs (unpublished data and reference 26).
Mentions: To explore the effect of inflammatory cytokines on LC differentiation and migration, we cultured LC-type cells in the presence of TNF-α. LCs may be derived from CD34+ hemopoietic progenitor-cells (45–48). However, these cells represent a wide range of maturation stages. To synchronize the maturation stages of LCs in the cultures we used mainly LC-type cells derived from human monocytes, grown in the presence of GM-CSF, IL-4, and TGF-β1 (21, 41). This further allowed us to compare LCs with DCi derived from the same monocytes differentiated in the presence of GM-CSF and IL-4 (7, 21, 49). When cultured in the absence of TNF-α approximately one-third of the monocyte-derived LC-type cells expressed significant surface levels of Langerin (Fig. 3 A). Remarkably, the addition of TNF-α at days 5 or 6 of the culture strongly increased the proportion of Langerin+ cells (Fig. 3 A). Langerin+ cells increased from 33 ± 12% to 75 ± 15% of total cells (n = 6 independent experiments), with a considerable higher MFI (from 66 ± 20 to 250 ± 130). However, the presence of TGF-β1 was mandatory for the induction of Langerin and BGs by TNF-α (unpublished data) demonstrating that TNF-α acts in synergy with TGF-β1 to promote LC differentiation.

Bottom Line: This indicates that LC migration and maturation can be independently regulated events.We suggest that during DL, inflammatory stimuli in the skin increase the migration of LCs to the lymph node but without associated maturation.Immature LCs might regulate immune responses during chronic inflammation.

View Article: PubMed Central - PubMed

Affiliation: Service d'Anatomie Pathologique, IFR Necker-Enfants Malades, 75015 Paris, France. geissman@necker.fr

ABSTRACT
The coordinated migration and maturation of dendritic cells (DCs) such as intraepithelial Langerhans cells (LCs) is considered critical for T cell priming in response to inflammation in the periphery. However, little is known about the role of inflammatory mediators for LC maturation and recruitment to lymph nodes in vivo. Here we show in human dermatopathic lymphadenitis (DL), which features an expanded population of LCs in one draining lymph node associated with inflammatory lesions in its tributary skin area, that the Langerin/CD207(+) LCs constitute a predominant population of immature DCs, which express CD1a, and CD68, but not CD83, CD86, and DC-lysosomal-associated membrane protein (LAMP)/CD208. Using LC-type cells generated in vitro in the presence of transforming growth factor (TGF)-beta1, we further found that tumor necrosis factor (TNF)-alpha, as a prototype proinflammatory factor, and a variety of inflammatory stimuli and bacterial products, increase Langerin expression and Langerin dependent Birbeck granules formation in cell which nevertheless lack costimulatory molecules, DC-LAMP/CD208 and potent T cell stimulatory activity but express CCR7 and respond to the lymph node homing chemokines CCL19 and CCL21. This indicates that LC migration and maturation can be independently regulated events. We suggest that during DL, inflammatory stimuli in the skin increase the migration of LCs to the lymph node but without associated maturation. Immature LCs might regulate immune responses during chronic inflammation.

Show MeSH
Related in: MedlinePlus