Limits...
Crucial role of interferon consensus sequence binding protein, but neither of interferon regulatory factor 1 nor of nitric oxide synthesis for protection against murine listeriosis.

Fehr T, Schoedon G, Odermatt B, Holtschke T, Schneemann M, Bachmann MF, Mak TW, Horak I, Zinkernagel RM - J. Exp. Med. (1997)

Bottom Line: However, the involved signaling pathways and effector mechanisms are still poorly understood.This correlated with impaired elimination of Listeria from infected peritoneal macrophage (PEM) cultures stimulated with IFN-gamma in vitro; in addition these cultures showed reduced and delayed oxidative burst upon IFN-gamma stimulation, whereas nitric oxide production was normal.These results indicate that (a) the ICSBP/IRF2 complex is essential for IFN-gamma-mediated protection against Listeria and that (b) ROI together with additional still unknown effector mechanisms may be responsible for the anti-Listeria activity of macrophages, whereas IRF1-induced RNI are not limiting.

View Article: PubMed Central - PubMed

Affiliation: Institute of Experimental Immunology, University Hospital, Zürich, Switzerland.

ABSTRACT
Listeria monocytogenes is widely used as a model to study immune responses against intracellular bacteria. It has been shown that neutrophils and macrophages play an important role to restrict bacterial replication in the early phase of primary infection in mice, and that the cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) are essential for protection. However, the involved signaling pathways and effector mechanisms are still poorly understood. This study investigated mouse strains deficient for the IFN-dependent transcription factors interferon consensus sequence binding protein (ICSBP), interferon regulatory factor (IRF) 1 or 2 for their capacity to eliminate Listeria in vivo and in vitro and for production of inducible reactive nitrogen intermediates (RNI) or reactive oxygen intermediates (ROI) in macrophages. ICSBP-/- and to a lesser degree also IRF2-/- mice were highly susceptible to Listeria infection. This correlated with impaired elimination of Listeria from infected peritoneal macrophage (PEM) cultures stimulated with IFN-gamma in vitro; in addition these cultures showed reduced and delayed oxidative burst upon IFN-gamma stimulation, whereas nitric oxide production was normal. In contrast, mice deficient for IRF1 were not able to produce nitric oxide, but they efficiently controlled Listeria in vivo and in vitro. These results indicate that (a) the ICSBP/IRF2 complex is essential for IFN-gamma-mediated protection against Listeria and that (b) ROI together with additional still unknown effector mechanisms may be responsible for the anti-Listeria activity of macrophages, whereas IRF1-induced RNI are not limiting.

Show MeSH

Related in: MedlinePlus

NO production and respiratory burst in PEM cultures. PEMs  of mice deficient for different IFN-related transcription factors were cultured as described in Fig. 3. (A) After 42 h NO production was measured  by determination of nitrite accumulation in the culture supernatants by  using Griess reagent. Values are calculated as nmol nitrite per 105 cells.  One of three independent and comparable experiments is shown. (B)  Respiratory burst capacity of the same PEMs was determined by measuring H2O2 production by chemiluminescence after stimulation with PMA.  Values were first calculated as nmol H2O2 per 105 cells, and then a stimulation index of stimulated versus unstimulated cultures was determined  (stimulation index of unstimulated culture = 1). Error bars indicate standard deviation.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196174&req=5

Figure 4: NO production and respiratory burst in PEM cultures. PEMs of mice deficient for different IFN-related transcription factors were cultured as described in Fig. 3. (A) After 42 h NO production was measured by determination of nitrite accumulation in the culture supernatants by using Griess reagent. Values are calculated as nmol nitrite per 105 cells. One of three independent and comparable experiments is shown. (B) Respiratory burst capacity of the same PEMs was determined by measuring H2O2 production by chemiluminescence after stimulation with PMA. Values were first calculated as nmol H2O2 per 105 cells, and then a stimulation index of stimulated versus unstimulated cultures was determined (stimulation index of unstimulated culture = 1). Error bars indicate standard deviation.

Mentions: Respiratory burst was measured as H2O2 production by cultured PEM upon PMA (Sigma, Buchs, Switzerland) stimulation as described (18). In brief, H2O2 secretion of macrophages was quantified by chemiluminescence under presence of horseradish peroxidase type I (Sigma) and 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol; Sigma) after triggering with 50 ng/ml PMA. Light emission was discontinuously measured over 15 min in a LKB 1251 luminometer (LKB, Bromma, Sweden). Values in mV were converted into pmol H2O2 after calibration by the scopoletin method (42). The cells on the cover slips were counted, and values for NO and H2O2 calculated as nmol/105 cells. In Fig. 4 B stimulation index of stimulated versus unstimulated cultures is shown, because absolute values of respiratory burst varied between the experiments. PMA was used to trigger respiratory burst because, as a chemically defined substance, it is the most reliable burst trigger. Also opsonized Listeria, BCG or zymosan could be used with similar capacities to trigger burst (23, 43), but more variability. Because we investigated mouse strains deficient for various IFNdependent transcription factors, the induction phase of NADPH oxidase during 2 d under IFN-γ stimulation is important in our experiment, whereas the effector phase of the burst trigger is only used as read-out to measure the enzyme activity by providing the best stimulator (PMA) and eccess of substrate (luminol).


Crucial role of interferon consensus sequence binding protein, but neither of interferon regulatory factor 1 nor of nitric oxide synthesis for protection against murine listeriosis.

Fehr T, Schoedon G, Odermatt B, Holtschke T, Schneemann M, Bachmann MF, Mak TW, Horak I, Zinkernagel RM - J. Exp. Med. (1997)

NO production and respiratory burst in PEM cultures. PEMs  of mice deficient for different IFN-related transcription factors were cultured as described in Fig. 3. (A) After 42 h NO production was measured  by determination of nitrite accumulation in the culture supernatants by  using Griess reagent. Values are calculated as nmol nitrite per 105 cells.  One of three independent and comparable experiments is shown. (B)  Respiratory burst capacity of the same PEMs was determined by measuring H2O2 production by chemiluminescence after stimulation with PMA.  Values were first calculated as nmol H2O2 per 105 cells, and then a stimulation index of stimulated versus unstimulated cultures was determined  (stimulation index of unstimulated culture = 1). Error bars indicate standard deviation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196174&req=5

Figure 4: NO production and respiratory burst in PEM cultures. PEMs of mice deficient for different IFN-related transcription factors were cultured as described in Fig. 3. (A) After 42 h NO production was measured by determination of nitrite accumulation in the culture supernatants by using Griess reagent. Values are calculated as nmol nitrite per 105 cells. One of three independent and comparable experiments is shown. (B) Respiratory burst capacity of the same PEMs was determined by measuring H2O2 production by chemiluminescence after stimulation with PMA. Values were first calculated as nmol H2O2 per 105 cells, and then a stimulation index of stimulated versus unstimulated cultures was determined (stimulation index of unstimulated culture = 1). Error bars indicate standard deviation.
Mentions: Respiratory burst was measured as H2O2 production by cultured PEM upon PMA (Sigma, Buchs, Switzerland) stimulation as described (18). In brief, H2O2 secretion of macrophages was quantified by chemiluminescence under presence of horseradish peroxidase type I (Sigma) and 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol; Sigma) after triggering with 50 ng/ml PMA. Light emission was discontinuously measured over 15 min in a LKB 1251 luminometer (LKB, Bromma, Sweden). Values in mV were converted into pmol H2O2 after calibration by the scopoletin method (42). The cells on the cover slips were counted, and values for NO and H2O2 calculated as nmol/105 cells. In Fig. 4 B stimulation index of stimulated versus unstimulated cultures is shown, because absolute values of respiratory burst varied between the experiments. PMA was used to trigger respiratory burst because, as a chemically defined substance, it is the most reliable burst trigger. Also opsonized Listeria, BCG or zymosan could be used with similar capacities to trigger burst (23, 43), but more variability. Because we investigated mouse strains deficient for various IFNdependent transcription factors, the induction phase of NADPH oxidase during 2 d under IFN-γ stimulation is important in our experiment, whereas the effector phase of the burst trigger is only used as read-out to measure the enzyme activity by providing the best stimulator (PMA) and eccess of substrate (luminol).

Bottom Line: However, the involved signaling pathways and effector mechanisms are still poorly understood.This correlated with impaired elimination of Listeria from infected peritoneal macrophage (PEM) cultures stimulated with IFN-gamma in vitro; in addition these cultures showed reduced and delayed oxidative burst upon IFN-gamma stimulation, whereas nitric oxide production was normal.These results indicate that (a) the ICSBP/IRF2 complex is essential for IFN-gamma-mediated protection against Listeria and that (b) ROI together with additional still unknown effector mechanisms may be responsible for the anti-Listeria activity of macrophages, whereas IRF1-induced RNI are not limiting.

View Article: PubMed Central - PubMed

Affiliation: Institute of Experimental Immunology, University Hospital, Zürich, Switzerland.

ABSTRACT
Listeria monocytogenes is widely used as a model to study immune responses against intracellular bacteria. It has been shown that neutrophils and macrophages play an important role to restrict bacterial replication in the early phase of primary infection in mice, and that the cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) are essential for protection. However, the involved signaling pathways and effector mechanisms are still poorly understood. This study investigated mouse strains deficient for the IFN-dependent transcription factors interferon consensus sequence binding protein (ICSBP), interferon regulatory factor (IRF) 1 or 2 for their capacity to eliminate Listeria in vivo and in vitro and for production of inducible reactive nitrogen intermediates (RNI) or reactive oxygen intermediates (ROI) in macrophages. ICSBP-/- and to a lesser degree also IRF2-/- mice were highly susceptible to Listeria infection. This correlated with impaired elimination of Listeria from infected peritoneal macrophage (PEM) cultures stimulated with IFN-gamma in vitro; in addition these cultures showed reduced and delayed oxidative burst upon IFN-gamma stimulation, whereas nitric oxide production was normal. In contrast, mice deficient for IRF1 were not able to produce nitric oxide, but they efficiently controlled Listeria in vivo and in vitro. These results indicate that (a) the ICSBP/IRF2 complex is essential for IFN-gamma-mediated protection against Listeria and that (b) ROI together with additional still unknown effector mechanisms may be responsible for the anti-Listeria activity of macrophages, whereas IRF1-induced RNI are not limiting.

Show MeSH
Related in: MedlinePlus