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Fibroblasts as host cells in latent leishmaniosis.

Bogdan C, Donhauser N, Döring R, Röllinghoff M, Diefenbach A, Rittig MG - J. Exp. Med. (2000)

Bottom Line: Intracellular parasites are known to persist lifelong in mammalian hosts after the clinical cure of the disease, but the mechanisms of persistence are poorly understood.In vitro, both promastigotes and amastigotes of L. major infected primary skin or lymph node fibroblasts.Compared with macrophages, cytokine-activated fibroblasts had a reduced ability to express type 2 nitric oxide synthase and to kill intracellular L. major.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Microbiology, Department of Anatomy, University of Erlangen, D-91054 Erlangen, Germany. christian.bogdan@mikrobio.med.uni-erlangen.de

ABSTRACT
Intracellular parasites are known to persist lifelong in mammalian hosts after the clinical cure of the disease, but the mechanisms of persistence are poorly understood. Here, we show by confocal laser microscopy that in the draining lymph nodes of mice that had healed a cutaneous infection with Leishmania major, 40% of the persisting parasites were associated with fibroblasts forming the reticular meshwork of the lymph nodes. In vitro, both promastigotes and amastigotes of L. major infected primary skin or lymph node fibroblasts. Compared with macrophages, cytokine-activated fibroblasts had a reduced ability to express type 2 nitric oxide synthase and to kill intracellular L. major. These data identify fibroblasts as an important host cell for Leishmania during the chronic phase of infection and suggest that they might serve as safe targets for the parasites in clinically latent disease.

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Expression of fibroblast markers by NOBO-1 cells (reticular fibroblasts isolated from the lymph node of a C57BL/6 mouse at day 239 of infection with L. major; A–D), RPMs (E–H), or thioglycollate-elicited macrophages (J–M). The cells were kept in cultures for 4–6 d and then analyzed for the expression of fibulin-2 (A, E, and J), perlecan (B, F, and K), or ER-TR7 (C, G, and L) by immunoperoxidase staining and hematoxylin counterstaining. Results similar to those shown for NOBO-1 cells were also obtained with primary skin fibroblasts of C57BL/6 mice (not shown). For control purposes, the primary antibodies were omitted and the cells were incubated with the biotinylated secondary reagent alone followed by streptavidin-peroxidase (D, H, and M). Original magnifications: ×1,000.
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Figure 1: Expression of fibroblast markers by NOBO-1 cells (reticular fibroblasts isolated from the lymph node of a C57BL/6 mouse at day 239 of infection with L. major; A–D), RPMs (E–H), or thioglycollate-elicited macrophages (J–M). The cells were kept in cultures for 4–6 d and then analyzed for the expression of fibulin-2 (A, E, and J), perlecan (B, F, and K), or ER-TR7 (C, G, and L) by immunoperoxidase staining and hematoxylin counterstaining. Results similar to those shown for NOBO-1 cells were also obtained with primary skin fibroblasts of C57BL/6 mice (not shown). For control purposes, the primary antibodies were omitted and the cells were incubated with the biotinylated secondary reagent alone followed by streptavidin-peroxidase (D, H, and M). Original magnifications: ×1,000.

Mentions: Fibroblasts were isolated from the skin of naive mice and from the draining (popliteal) lymph nodes of mice that had healed a cutaneous infection with L. major (>day 200 after infection). Three skin fibroblast lines (CHF-1, CHF-2, and NOSS-1) and two lymph node reticular fibroblast lines (NOBO-1 and NOBO-3) were established and used for in vitro experiments at passage numbers <25. The cells were determined to be fibroblasts on the following grounds: (a) characteristic morphology, i.e., formation of monolayers consisting of cells that assumed a pavement-like and spindle-shaped appearance when confluent (see Fig. 2); (b) serum-dependent growth (data not shown); (c) absence of markers characteristic for macrophages (F4/80, Mac-1, BM-8, ER-MP-23), dendritic cells (NLDC-145, MHC class II), granulocytes (GR-1), T cells (Thy 1.2), B cells (B220), or endothelial cells (MECA-32) (data not shown); (d) production of a variety of known matrix proteins (fibronectin, laminin-1, fibulin-2, perlecan, and collagen VI) during in vitro culture for 1–6 d (Fig. 1a and Fig. b, and data not shown); (e) positive staining with the rat mAb ER-TR7 (Fig. 1 C, and data not shown), which detects an intracellular component of mouse reticular fibroblasts but also reacts with an extracellular connective tissue product that is different from the matrix proteins laminin, fibronectin, types I–V collagen, heparan sulfate proteoglycan, entactin, and nidogen 33. RPMs or PEMs, in contrast, were negative for perlecan, fibulin-2, collagen VI, and ER-TR7 throughout a culture period of 6 d (Fig. 1eFig. fFig. gFig. hFig. jFig. kFig. lFig. m) and showed only a very weak and transient staining with antilaminin or antifibronectin antiserum (not shown). Thus, ER-TR7, perlecan, and fibulin-2 (and in some experiments also laminin, fibronectin, and collagen VI) were used as primary markers for the detection of fibroblasts in lymph nodes.


Fibroblasts as host cells in latent leishmaniosis.

Bogdan C, Donhauser N, Döring R, Röllinghoff M, Diefenbach A, Rittig MG - J. Exp. Med. (2000)

Expression of fibroblast markers by NOBO-1 cells (reticular fibroblasts isolated from the lymph node of a C57BL/6 mouse at day 239 of infection with L. major; A–D), RPMs (E–H), or thioglycollate-elicited macrophages (J–M). The cells were kept in cultures for 4–6 d and then analyzed for the expression of fibulin-2 (A, E, and J), perlecan (B, F, and K), or ER-TR7 (C, G, and L) by immunoperoxidase staining and hematoxylin counterstaining. Results similar to those shown for NOBO-1 cells were also obtained with primary skin fibroblasts of C57BL/6 mice (not shown). For control purposes, the primary antibodies were omitted and the cells were incubated with the biotinylated secondary reagent alone followed by streptavidin-peroxidase (D, H, and M). Original magnifications: ×1,000.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193203&req=5

Figure 1: Expression of fibroblast markers by NOBO-1 cells (reticular fibroblasts isolated from the lymph node of a C57BL/6 mouse at day 239 of infection with L. major; A–D), RPMs (E–H), or thioglycollate-elicited macrophages (J–M). The cells were kept in cultures for 4–6 d and then analyzed for the expression of fibulin-2 (A, E, and J), perlecan (B, F, and K), or ER-TR7 (C, G, and L) by immunoperoxidase staining and hematoxylin counterstaining. Results similar to those shown for NOBO-1 cells were also obtained with primary skin fibroblasts of C57BL/6 mice (not shown). For control purposes, the primary antibodies were omitted and the cells were incubated with the biotinylated secondary reagent alone followed by streptavidin-peroxidase (D, H, and M). Original magnifications: ×1,000.
Mentions: Fibroblasts were isolated from the skin of naive mice and from the draining (popliteal) lymph nodes of mice that had healed a cutaneous infection with L. major (>day 200 after infection). Three skin fibroblast lines (CHF-1, CHF-2, and NOSS-1) and two lymph node reticular fibroblast lines (NOBO-1 and NOBO-3) were established and used for in vitro experiments at passage numbers <25. The cells were determined to be fibroblasts on the following grounds: (a) characteristic morphology, i.e., formation of monolayers consisting of cells that assumed a pavement-like and spindle-shaped appearance when confluent (see Fig. 2); (b) serum-dependent growth (data not shown); (c) absence of markers characteristic for macrophages (F4/80, Mac-1, BM-8, ER-MP-23), dendritic cells (NLDC-145, MHC class II), granulocytes (GR-1), T cells (Thy 1.2), B cells (B220), or endothelial cells (MECA-32) (data not shown); (d) production of a variety of known matrix proteins (fibronectin, laminin-1, fibulin-2, perlecan, and collagen VI) during in vitro culture for 1–6 d (Fig. 1a and Fig. b, and data not shown); (e) positive staining with the rat mAb ER-TR7 (Fig. 1 C, and data not shown), which detects an intracellular component of mouse reticular fibroblasts but also reacts with an extracellular connective tissue product that is different from the matrix proteins laminin, fibronectin, types I–V collagen, heparan sulfate proteoglycan, entactin, and nidogen 33. RPMs or PEMs, in contrast, were negative for perlecan, fibulin-2, collagen VI, and ER-TR7 throughout a culture period of 6 d (Fig. 1eFig. fFig. gFig. hFig. jFig. kFig. lFig. m) and showed only a very weak and transient staining with antilaminin or antifibronectin antiserum (not shown). Thus, ER-TR7, perlecan, and fibulin-2 (and in some experiments also laminin, fibronectin, and collagen VI) were used as primary markers for the detection of fibroblasts in lymph nodes.

Bottom Line: Intracellular parasites are known to persist lifelong in mammalian hosts after the clinical cure of the disease, but the mechanisms of persistence are poorly understood.In vitro, both promastigotes and amastigotes of L. major infected primary skin or lymph node fibroblasts.Compared with macrophages, cytokine-activated fibroblasts had a reduced ability to express type 2 nitric oxide synthase and to kill intracellular L. major.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Microbiology, Department of Anatomy, University of Erlangen, D-91054 Erlangen, Germany. christian.bogdan@mikrobio.med.uni-erlangen.de

ABSTRACT
Intracellular parasites are known to persist lifelong in mammalian hosts after the clinical cure of the disease, but the mechanisms of persistence are poorly understood. Here, we show by confocal laser microscopy that in the draining lymph nodes of mice that had healed a cutaneous infection with Leishmania major, 40% of the persisting parasites were associated with fibroblasts forming the reticular meshwork of the lymph nodes. In vitro, both promastigotes and amastigotes of L. major infected primary skin or lymph node fibroblasts. Compared with macrophages, cytokine-activated fibroblasts had a reduced ability to express type 2 nitric oxide synthase and to kill intracellular L. major. These data identify fibroblasts as an important host cell for Leishmania during the chronic phase of infection and suggest that they might serve as safe targets for the parasites in clinically latent disease.

Show MeSH
Related in: MedlinePlus