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Development of inflammatory angiogenesis by local stimulation of Fas in vivo.

Biancone L, Martino AD, Orlandi V, Conaldi PG, Toniolo A, Camussi G - J. Exp. Med. (1997)

Bottom Line: Several mast cells were also detected in the inflammatory infiltrate.The phenomenon was dose and time dependent and required the presence of heparin.Apoptotic cells were not detectable at any time inside the implant or in the surrounding tissue, suggesting that angiogenesis and cell infiltration did not result from recruitment of phagocytes by apoptotic cells but rather by a stimulatory signal through Fas-engagement.

View Article: PubMed Central - PubMed

Affiliation: Chair of Nephrology, Department of Clinical and Biological Sciences, University of Pavia, Varese, Italy.

ABSTRACT
Fas-Fas ligand interaction is thought to be a crucial mechanism in controlling lymphocyte expansion by inducing lymphocyte apoptosis. However, Fas is also broadly expressed on nonlymphoid cells, where its function in vivo remains to be determined. In this study, we describe the development of inflammatory angiogenesis induced by agonistic anti-Fas mAb Jo2 in a murine model where Matrigel is used as a vehicle for the delivery of mediators. The subcutaneous implants in mice of Matrigel containing mAb Jo2 became rapidly infiltrated by endothelial cells and by scattered monocytes and macrophages. After formation and canalization of new vessels, marked intravascular accumulation and extravasation of neutrophils were observed. Several mast cells were also detected in the inflammatory infiltrate. The phenomenon was dose and time dependent and required the presence of heparin. The dependency on activation of Fas is suggested by the observation that the inflammatory angiogenesis was restricted to the agonistic anti-Fas mAb and it was absent in lpr Fas-mutant mice. Apoptotic cells were not detectable at any time inside the implant or in the surrounding tissue, suggesting that angiogenesis and cell infiltration did not result from recruitment of phagocytes by apoptotic cells but rather by a stimulatory signal through Fas-engagement. These findings suggest a role for Fas-Fas ligand interaction in promoting local angiogenesis and inflammation.

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Histological and immunohistochemical analysis of  Matrigel implants. Representative hematoxylin-eosin–stained  section of Matrigel containing 5  μg/ml anti-Fas mAb Jo2 (A) implanted in wild-type mice and  excised 6 d after injection (6  mice per condition in each experiment). Progressive invasion  of cells into the Matrigel implant  and formation of capillaries is  observed (×200). (B) Control:  mAb Jo2 was replaced by hamster IgG (×200). Similar results  were obtained by using nonagonistic anti-Fas mAb RMF6 or by  injecting Matrigel with mAb  Jo2 into lpr Fas-mutant mice (not  shown). Kinetic of anti-Fas mAb  Jo2-induced inflammatory angiogenesis (×400); (C) Initial  migration of endothelial cells  aligning to form cord-like structures at day 1; (D) appearance of  PMN inside the lumen of neocapillaries (day 2); (E) marked  extravasation of PMN throughout the neoformed capillary into  the Matrigel (day 6); (F) metachromatic staining by Toluidine blue  of mast cells infiltrating Matrigel  at day 6 (×600); (G) formation  of canalized vessels containing  erythrocytes and leukocytes (day  4–6). Immunohistochemical detection of apoptotic cells by the  TUNEL method (normal nuclei  are counterstained with propidium iodide in red); (H) Section  of Matrigel with anti-Fas mAb  Jo2 at day 6, displaying total absence of apoptotic cells among  the infiltrating cells (×200). The  same results were obtained on  Matrigel excised 6 h, 12 h, 24 h,  5 d, and 10 d after injection (not  shown); (I) Rat regressing mammary glands obtained at the fourth  day after weaning showing apoptotic cells (arrows) along the acini,  as positive control (×200).
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Figure 1: Histological and immunohistochemical analysis of Matrigel implants. Representative hematoxylin-eosin–stained section of Matrigel containing 5 μg/ml anti-Fas mAb Jo2 (A) implanted in wild-type mice and excised 6 d after injection (6 mice per condition in each experiment). Progressive invasion of cells into the Matrigel implant and formation of capillaries is observed (×200). (B) Control: mAb Jo2 was replaced by hamster IgG (×200). Similar results were obtained by using nonagonistic anti-Fas mAb RMF6 or by injecting Matrigel with mAb Jo2 into lpr Fas-mutant mice (not shown). Kinetic of anti-Fas mAb Jo2-induced inflammatory angiogenesis (×400); (C) Initial migration of endothelial cells aligning to form cord-like structures at day 1; (D) appearance of PMN inside the lumen of neocapillaries (day 2); (E) marked extravasation of PMN throughout the neoformed capillary into the Matrigel (day 6); (F) metachromatic staining by Toluidine blue of mast cells infiltrating Matrigel at day 6 (×600); (G) formation of canalized vessels containing erythrocytes and leukocytes (day 4–6). Immunohistochemical detection of apoptotic cells by the TUNEL method (normal nuclei are counterstained with propidium iodide in red); (H) Section of Matrigel with anti-Fas mAb Jo2 at day 6, displaying total absence of apoptotic cells among the infiltrating cells (×200). The same results were obtained on Matrigel excised 6 h, 12 h, 24 h, 5 d, and 10 d after injection (not shown); (I) Rat regressing mammary glands obtained at the fourth day after weaning showing apoptotic cells (arrows) along the acini, as positive control (×200).

Mentions: Subcutaneous injection in mice of Matrigel containing the agonistic anti-Fas mAb Jo2 caused rapid neovascularization and infiltration of inflammatory cells within the implant (Fig. 1). The phenomenon is dose and time-dependent (Figs. 2 and 3). The minimal effective dose was 100 ng/ml. Initial infiltration of cells was observed as early as 24 h after injection (Fig. 2 A) and the infiltrating cells were mainly endothelial cells (Fig. 1 A), as detected by immunofluorescence staining for vWF (Fig. 4, A and B) and MAC-1+ (Fig. 4 C) and aspecific esterase plus monocytes. Polymorphonuclear neutrophils (PMN) were seen initially at the periphery of Matrigel implant, then within the lumen of neoformed capillary sprouts (Fig. 1 D) and finally around the neoformed vessels (Fig. 1 F). Only scattered lymphocytes CD4+ or CD8+ were detected. Several mast cells (Fig. 1 F), metachromatically stained by Toluidine blue, were also detected within the Matrigel and the inflammatory infiltrate surrounding the neoformed vessels. Maximal angiogenesis was observed at day 6 with formation of canalized vessels (Fig. 1 G and 3 A). At that time, ∼50% of neoformed vessels were surrounded by an intense infiltration of inflammatory cells similar to granulation-like tissue (Fig. 1 E). Control purified hamster IgG and a nonsignaling anti–mouse Fas mAb RMF6 (17) were completely ineffective (Fig. 5). mAb Jo2 was endotoxin free as tested by Limulus assay and in selected experiments preincubation for 30 min of the antibody with 5 μg/ml polymyxin B, which complexes and inactivates endotoxin, did not abrogate the inflammatory angiogenesis induced by this mAb (data not shown). All the animals that were implanted with Matrigel containing mAb Jo2 remained apparently healthy and active and the histological examination of the liver did not show macroscopic alterations.


Development of inflammatory angiogenesis by local stimulation of Fas in vivo.

Biancone L, Martino AD, Orlandi V, Conaldi PG, Toniolo A, Camussi G - J. Exp. Med. (1997)

Histological and immunohistochemical analysis of  Matrigel implants. Representative hematoxylin-eosin–stained  section of Matrigel containing 5  μg/ml anti-Fas mAb Jo2 (A) implanted in wild-type mice and  excised 6 d after injection (6  mice per condition in each experiment). Progressive invasion  of cells into the Matrigel implant  and formation of capillaries is  observed (×200). (B) Control:  mAb Jo2 was replaced by hamster IgG (×200). Similar results  were obtained by using nonagonistic anti-Fas mAb RMF6 or by  injecting Matrigel with mAb  Jo2 into lpr Fas-mutant mice (not  shown). Kinetic of anti-Fas mAb  Jo2-induced inflammatory angiogenesis (×400); (C) Initial  migration of endothelial cells  aligning to form cord-like structures at day 1; (D) appearance of  PMN inside the lumen of neocapillaries (day 2); (E) marked  extravasation of PMN throughout the neoformed capillary into  the Matrigel (day 6); (F) metachromatic staining by Toluidine blue  of mast cells infiltrating Matrigel  at day 6 (×600); (G) formation  of canalized vessels containing  erythrocytes and leukocytes (day  4–6). Immunohistochemical detection of apoptotic cells by the  TUNEL method (normal nuclei  are counterstained with propidium iodide in red); (H) Section  of Matrigel with anti-Fas mAb  Jo2 at day 6, displaying total absence of apoptotic cells among  the infiltrating cells (×200). The  same results were obtained on  Matrigel excised 6 h, 12 h, 24 h,  5 d, and 10 d after injection (not  shown); (I) Rat regressing mammary glands obtained at the fourth  day after weaning showing apoptotic cells (arrows) along the acini,  as positive control (×200).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198950&req=5

Figure 1: Histological and immunohistochemical analysis of Matrigel implants. Representative hematoxylin-eosin–stained section of Matrigel containing 5 μg/ml anti-Fas mAb Jo2 (A) implanted in wild-type mice and excised 6 d after injection (6 mice per condition in each experiment). Progressive invasion of cells into the Matrigel implant and formation of capillaries is observed (×200). (B) Control: mAb Jo2 was replaced by hamster IgG (×200). Similar results were obtained by using nonagonistic anti-Fas mAb RMF6 or by injecting Matrigel with mAb Jo2 into lpr Fas-mutant mice (not shown). Kinetic of anti-Fas mAb Jo2-induced inflammatory angiogenesis (×400); (C) Initial migration of endothelial cells aligning to form cord-like structures at day 1; (D) appearance of PMN inside the lumen of neocapillaries (day 2); (E) marked extravasation of PMN throughout the neoformed capillary into the Matrigel (day 6); (F) metachromatic staining by Toluidine blue of mast cells infiltrating Matrigel at day 6 (×600); (G) formation of canalized vessels containing erythrocytes and leukocytes (day 4–6). Immunohistochemical detection of apoptotic cells by the TUNEL method (normal nuclei are counterstained with propidium iodide in red); (H) Section of Matrigel with anti-Fas mAb Jo2 at day 6, displaying total absence of apoptotic cells among the infiltrating cells (×200). The same results were obtained on Matrigel excised 6 h, 12 h, 24 h, 5 d, and 10 d after injection (not shown); (I) Rat regressing mammary glands obtained at the fourth day after weaning showing apoptotic cells (arrows) along the acini, as positive control (×200).
Mentions: Subcutaneous injection in mice of Matrigel containing the agonistic anti-Fas mAb Jo2 caused rapid neovascularization and infiltration of inflammatory cells within the implant (Fig. 1). The phenomenon is dose and time-dependent (Figs. 2 and 3). The minimal effective dose was 100 ng/ml. Initial infiltration of cells was observed as early as 24 h after injection (Fig. 2 A) and the infiltrating cells were mainly endothelial cells (Fig. 1 A), as detected by immunofluorescence staining for vWF (Fig. 4, A and B) and MAC-1+ (Fig. 4 C) and aspecific esterase plus monocytes. Polymorphonuclear neutrophils (PMN) were seen initially at the periphery of Matrigel implant, then within the lumen of neoformed capillary sprouts (Fig. 1 D) and finally around the neoformed vessels (Fig. 1 F). Only scattered lymphocytes CD4+ or CD8+ were detected. Several mast cells (Fig. 1 F), metachromatically stained by Toluidine blue, were also detected within the Matrigel and the inflammatory infiltrate surrounding the neoformed vessels. Maximal angiogenesis was observed at day 6 with formation of canalized vessels (Fig. 1 G and 3 A). At that time, ∼50% of neoformed vessels were surrounded by an intense infiltration of inflammatory cells similar to granulation-like tissue (Fig. 1 E). Control purified hamster IgG and a nonsignaling anti–mouse Fas mAb RMF6 (17) were completely ineffective (Fig. 5). mAb Jo2 was endotoxin free as tested by Limulus assay and in selected experiments preincubation for 30 min of the antibody with 5 μg/ml polymyxin B, which complexes and inactivates endotoxin, did not abrogate the inflammatory angiogenesis induced by this mAb (data not shown). All the animals that were implanted with Matrigel containing mAb Jo2 remained apparently healthy and active and the histological examination of the liver did not show macroscopic alterations.

Bottom Line: Several mast cells were also detected in the inflammatory infiltrate.The phenomenon was dose and time dependent and required the presence of heparin.Apoptotic cells were not detectable at any time inside the implant or in the surrounding tissue, suggesting that angiogenesis and cell infiltration did not result from recruitment of phagocytes by apoptotic cells but rather by a stimulatory signal through Fas-engagement.

View Article: PubMed Central - PubMed

Affiliation: Chair of Nephrology, Department of Clinical and Biological Sciences, University of Pavia, Varese, Italy.

ABSTRACT
Fas-Fas ligand interaction is thought to be a crucial mechanism in controlling lymphocyte expansion by inducing lymphocyte apoptosis. However, Fas is also broadly expressed on nonlymphoid cells, where its function in vivo remains to be determined. In this study, we describe the development of inflammatory angiogenesis induced by agonistic anti-Fas mAb Jo2 in a murine model where Matrigel is used as a vehicle for the delivery of mediators. The subcutaneous implants in mice of Matrigel containing mAb Jo2 became rapidly infiltrated by endothelial cells and by scattered monocytes and macrophages. After formation and canalization of new vessels, marked intravascular accumulation and extravasation of neutrophils were observed. Several mast cells were also detected in the inflammatory infiltrate. The phenomenon was dose and time dependent and required the presence of heparin. The dependency on activation of Fas is suggested by the observation that the inflammatory angiogenesis was restricted to the agonistic anti-Fas mAb and it was absent in lpr Fas-mutant mice. Apoptotic cells were not detectable at any time inside the implant or in the surrounding tissue, suggesting that angiogenesis and cell infiltration did not result from recruitment of phagocytes by apoptotic cells but rather by a stimulatory signal through Fas-engagement. These findings suggest a role for Fas-Fas ligand interaction in promoting local angiogenesis and inflammation.

Show MeSH
Related in: MedlinePlus