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Opposing effects of transmembrane and soluble Fas ligand expression on inflammation and tumor cell survival.

Hohlbaum AM, Moe S, Marshak-Rothstein A - J. Exp. Med. (2000)

Bottom Line: To rigorously assess the physiological role of different forms of the FasL molecule with regard to these two distinct processes, we isolated stably transfected lymphoma cell lines that expressed either murine wild-type FasL, membrane-only FasL, or functionally distinct forms of soluble FasL.Our study clearly demonstrated that the extent of inflammation induced by the transfectants directly correlated with their relative cytotoxic activities.However, expression of soluble FasL was not benign, but actually suppressed the inflammatory response and protected other transfectants from the effector mechanisms elicted by membrane-bound FasL.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118, USA.

ABSTRACT
Fas ligand (FasL) has been shown to mediate both apoptotic and inflammatory reactions. To rigorously assess the physiological role of different forms of the FasL molecule with regard to these two distinct processes, we isolated stably transfected lymphoma cell lines that expressed either murine wild-type FasL, membrane-only FasL, or functionally distinct forms of soluble FasL. First, the ability of these lines to induce an inflammatory response was assessed in vivo by injecting the transfectants intraperitoneally and measuring subsequent neutrophil extravasation into the peritoneal cavity. Second, lines were assessed by injecting the transfectants subcutaneously and monitoring their growth as solid tumors. Our study clearly demonstrated that the extent of inflammation induced by the transfectants directly correlated with their relative cytotoxic activities. A neutrophil response could only be elicited in mice with intact Fas death domains although Fas expression by the neutrophils was not essential. Lymphoma cells expressing the soluble FasL form corresponding to the natural cleavage product could not trigger apoptosis and did not induce a neutrophil response. In contrast to the other FasL transfectants, these cells survived as tumor transplants. However, expression of soluble FasL was not benign, but actually suppressed the inflammatory response and protected other transfectants from the effector mechanisms elicted by membrane-bound FasL.

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Inflammation depends on recipient death domain–intact Fas expression. (A) MRL-gld and MRL-lpr/gld mice were injected intraperitoneally with 5 × 106 L5-neo or L5-mFasL cells. After 16 h, PECs were prepared and analyzed as described in the legend to Fig. 3. Shown is one representative experiment out of two; bars represent the mean of three mice per group, and error bars represent the SD. (B) C3H.MRL-lpr, CBA-lprcg, and CBA/J mice were injected intraperitoneally with 5 × 106 L5-neo or L5-mFasL cells. After 16 h, PECs were prepared and analyzed as described in the legend to Fig. 3. The total number of neutrophils per mouse was compared between different groups. Data represent the mean of two mice per group, and error bars represent the SD. (C) Isolated thymocytes from C3H.MRL-lpr, CBA-lprcg, and CBA/J mice were stained for Fas surface expression with the mAb Jo-2. (D) The sensitivity of thymocytes from C3H.MRL-lpr, CBA-lprcg, and CBA/J mice to FasL-mediated killing was assessed. In brief, isolated thymocytes were 51Cr labeled and used as targets in a 6-h cytotoxic assay with L5-mFasL as effector cells. Target thymocytes were mixed with L5-mFasL cells at E/T ratios ranging from 12:1 to 0.3:1 and incubated for 6 h.
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Figure 4: Inflammation depends on recipient death domain–intact Fas expression. (A) MRL-gld and MRL-lpr/gld mice were injected intraperitoneally with 5 × 106 L5-neo or L5-mFasL cells. After 16 h, PECs were prepared and analyzed as described in the legend to Fig. 3. Shown is one representative experiment out of two; bars represent the mean of three mice per group, and error bars represent the SD. (B) C3H.MRL-lpr, CBA-lprcg, and CBA/J mice were injected intraperitoneally with 5 × 106 L5-neo or L5-mFasL cells. After 16 h, PECs were prepared and analyzed as described in the legend to Fig. 3. The total number of neutrophils per mouse was compared between different groups. Data represent the mean of two mice per group, and error bars represent the SD. (C) Isolated thymocytes from C3H.MRL-lpr, CBA-lprcg, and CBA/J mice were stained for Fas surface expression with the mAb Jo-2. (D) The sensitivity of thymocytes from C3H.MRL-lpr, CBA-lprcg, and CBA/J mice to FasL-mediated killing was assessed. In brief, isolated thymocytes were 51Cr labeled and used as targets in a 6-h cytotoxic assay with L5-mFasL as effector cells. Target thymocytes were mixed with L5-mFasL cells at E/T ratios ranging from 12:1 to 0.3:1 and incubated for 6 h.

Mentions: Although L5-neo cells were used as controls throughout the study, it was important to confirm that the observed effect was truly Fas dependent. MRL-gld and double mutant MRL-lpr/gld mice were inoculated intraperitoneally with 5 × 106 L5-neo or L5-mFasL cells. After 16 h, PECs were isolated and analyzed, as described in Fig. 3 C. Only mice with functional Fas expression (MRL-gld) were able to mount a neutrophil response. MRL-lpr/gld mice were completely resistant to high numbers of L5-mFasL cells (Fig. 4 A).


Opposing effects of transmembrane and soluble Fas ligand expression on inflammation and tumor cell survival.

Hohlbaum AM, Moe S, Marshak-Rothstein A - J. Exp. Med. (2000)

Inflammation depends on recipient death domain–intact Fas expression. (A) MRL-gld and MRL-lpr/gld mice were injected intraperitoneally with 5 × 106 L5-neo or L5-mFasL cells. After 16 h, PECs were prepared and analyzed as described in the legend to Fig. 3. Shown is one representative experiment out of two; bars represent the mean of three mice per group, and error bars represent the SD. (B) C3H.MRL-lpr, CBA-lprcg, and CBA/J mice were injected intraperitoneally with 5 × 106 L5-neo or L5-mFasL cells. After 16 h, PECs were prepared and analyzed as described in the legend to Fig. 3. The total number of neutrophils per mouse was compared between different groups. Data represent the mean of two mice per group, and error bars represent the SD. (C) Isolated thymocytes from C3H.MRL-lpr, CBA-lprcg, and CBA/J mice were stained for Fas surface expression with the mAb Jo-2. (D) The sensitivity of thymocytes from C3H.MRL-lpr, CBA-lprcg, and CBA/J mice to FasL-mediated killing was assessed. In brief, isolated thymocytes were 51Cr labeled and used as targets in a 6-h cytotoxic assay with L5-mFasL as effector cells. Target thymocytes were mixed with L5-mFasL cells at E/T ratios ranging from 12:1 to 0.3:1 and incubated for 6 h.
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Related In: Results  -  Collection

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Figure 4: Inflammation depends on recipient death domain–intact Fas expression. (A) MRL-gld and MRL-lpr/gld mice were injected intraperitoneally with 5 × 106 L5-neo or L5-mFasL cells. After 16 h, PECs were prepared and analyzed as described in the legend to Fig. 3. Shown is one representative experiment out of two; bars represent the mean of three mice per group, and error bars represent the SD. (B) C3H.MRL-lpr, CBA-lprcg, and CBA/J mice were injected intraperitoneally with 5 × 106 L5-neo or L5-mFasL cells. After 16 h, PECs were prepared and analyzed as described in the legend to Fig. 3. The total number of neutrophils per mouse was compared between different groups. Data represent the mean of two mice per group, and error bars represent the SD. (C) Isolated thymocytes from C3H.MRL-lpr, CBA-lprcg, and CBA/J mice were stained for Fas surface expression with the mAb Jo-2. (D) The sensitivity of thymocytes from C3H.MRL-lpr, CBA-lprcg, and CBA/J mice to FasL-mediated killing was assessed. In brief, isolated thymocytes were 51Cr labeled and used as targets in a 6-h cytotoxic assay with L5-mFasL as effector cells. Target thymocytes were mixed with L5-mFasL cells at E/T ratios ranging from 12:1 to 0.3:1 and incubated for 6 h.
Mentions: Although L5-neo cells were used as controls throughout the study, it was important to confirm that the observed effect was truly Fas dependent. MRL-gld and double mutant MRL-lpr/gld mice were inoculated intraperitoneally with 5 × 106 L5-neo or L5-mFasL cells. After 16 h, PECs were isolated and analyzed, as described in Fig. 3 C. Only mice with functional Fas expression (MRL-gld) were able to mount a neutrophil response. MRL-lpr/gld mice were completely resistant to high numbers of L5-mFasL cells (Fig. 4 A).

Bottom Line: To rigorously assess the physiological role of different forms of the FasL molecule with regard to these two distinct processes, we isolated stably transfected lymphoma cell lines that expressed either murine wild-type FasL, membrane-only FasL, or functionally distinct forms of soluble FasL.Our study clearly demonstrated that the extent of inflammation induced by the transfectants directly correlated with their relative cytotoxic activities.However, expression of soluble FasL was not benign, but actually suppressed the inflammatory response and protected other transfectants from the effector mechanisms elicted by membrane-bound FasL.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118, USA.

ABSTRACT
Fas ligand (FasL) has been shown to mediate both apoptotic and inflammatory reactions. To rigorously assess the physiological role of different forms of the FasL molecule with regard to these two distinct processes, we isolated stably transfected lymphoma cell lines that expressed either murine wild-type FasL, membrane-only FasL, or functionally distinct forms of soluble FasL. First, the ability of these lines to induce an inflammatory response was assessed in vivo by injecting the transfectants intraperitoneally and measuring subsequent neutrophil extravasation into the peritoneal cavity. Second, lines were assessed by injecting the transfectants subcutaneously and monitoring their growth as solid tumors. Our study clearly demonstrated that the extent of inflammation induced by the transfectants directly correlated with their relative cytotoxic activities. A neutrophil response could only be elicited in mice with intact Fas death domains although Fas expression by the neutrophils was not essential. Lymphoma cells expressing the soluble FasL form corresponding to the natural cleavage product could not trigger apoptosis and did not induce a neutrophil response. In contrast to the other FasL transfectants, these cells survived as tumor transplants. However, expression of soluble FasL was not benign, but actually suppressed the inflammatory response and protected other transfectants from the effector mechanisms elicted by membrane-bound FasL.

Show MeSH
Related in: MedlinePlus