Limits...
Selective suppression of interleukin-12 induction after macrophage receptor ligation.

Sutterwala FS, Noel GJ, Clynes R, Mosser DM - J. Exp. Med. (1997)

Bottom Line: The results of several different experimental approaches suggest that IL-12 downregulation was due to extracellular calcium influxes that resulted from receptor ligation.Finally, bone marrow-derived macrophages from FcR gamma chain-deficient mice, which fail to flux calcium after receptor ligation, failed to inhibit IL-12(p40) mRNA induction.These results indicate that the calcium influxes that occur as a result of receptor ligation are responsible for inhibiting the induction of IL-12 by LPS.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.

ABSTRACT
Interleukin (IL)-12 is a monocyte- and macrophage-derived cytokine that plays a crucial role in both the innate and the acquired immune response. In this study, we examined the effects that ligating specific macrophage receptors had on the induction of IL-12 by lipopolysaccharide (LPS). We report that ligation of the macrophage Fcgamma, complement, or scavenger receptors inhibited the induction of IL-12 by LPS. Both mRNA synthesis and protein secretion were diminished to near-undetectable levels following receptor ligation. Suppression was specific to IL-12 since IL-10 and tumor necrosis factor-alpha (TNF-alpha) production were not inhibited by ligating macrophage receptors. The results of several different experimental approaches suggest that IL-12 downregulation was due to extracellular calcium influxes that resulted from receptor ligation. First, preventing extracellular calcium influxes, by performing the assays in EGTA, abrogated FcgammaR-mediated IL-12(p40) mRNA suppression. Second, exposure of macrophages to the calcium ionophores, ionomycin or A23187, mimicked receptor ligation and inhibited IL-12(p40) mRNA induction by LPS. Finally, bone marrow-derived macrophages from FcR gamma chain-deficient mice, which fail to flux calcium after receptor ligation, failed to inhibit IL-12(p40) mRNA induction. These results indicate that the calcium influxes that occur as a result of receptor ligation are responsible for inhibiting the induction of IL-12 by LPS. Hence, the ligation of phagocytic receptors on macrophages can lead to a dramatic decrease in IL-12 induction. This downregulation may be a way of limiting proinflammatory responses of macrophages to extracellular pathogens, or suppressing the development of cell-mediated immunity to intracellular pathogens.

Show MeSH

Related in: MedlinePlus

Effect of specific receptor ligation on the LPS- induced expression of cytokine  mRNA. (A) BMMφ from  BALB/c mice were exposed to  LPS (50 ng/ml), or LPS in combination with either E-IgG, E-C3bi,  E-ML-BSA, or latex microspheres. 6 h after the addition of  stimuli, total RNA was isolated  and used to carry out competitive RT-PCR, using primers for  HPRT, TNF-α, IL-10, or the  inducible p40 subunit of IL-12, as  described in Materials and Methods. (B) cDNA generated from  BALB/c BMMφ exposed to  LPS (50 ng/ml) or LPS in combination with E-IgG, were first  normalized for HPRT levels. Constant volumes of normalized cDNAs were then amplified in the presence of decreasing concentrations of competitor  (PQRS), using primers for the inducible p40 subunit of IL-12. The concentration of the experimental cDNA is represented by the equivalent intensities  of competitor and wild-type bands. The fold decrease in IL-12(p40) levels between BMMφ exposed to LPS or LPS in combination with E-IgG can be  determined by taking the ratio of their equivalence points.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196339&req=5

Figure 2: Effect of specific receptor ligation on the LPS- induced expression of cytokine mRNA. (A) BMMφ from BALB/c mice were exposed to LPS (50 ng/ml), or LPS in combination with either E-IgG, E-C3bi, E-ML-BSA, or latex microspheres. 6 h after the addition of stimuli, total RNA was isolated and used to carry out competitive RT-PCR, using primers for HPRT, TNF-α, IL-10, or the inducible p40 subunit of IL-12, as described in Materials and Methods. (B) cDNA generated from BALB/c BMMφ exposed to LPS (50 ng/ml) or LPS in combination with E-IgG, were first normalized for HPRT levels. Constant volumes of normalized cDNAs were then amplified in the presence of decreasing concentrations of competitor (PQRS), using primers for the inducible p40 subunit of IL-12. The concentration of the experimental cDNA is represented by the equivalent intensities of competitor and wild-type bands. The fold decrease in IL-12(p40) levels between BMMφ exposed to LPS or LPS in combination with E-IgG can be determined by taking the ratio of their equivalence points.

Mentions: BMMφ from BALB/c mice were incubated for 6 h with either LPS alone or simultaneously with LPS and particulate ligands (Fig. 2 A). LPS was a potent inducer of IL-12(p40), IL-10, and TNF-α mRNA. The ligation of Fcγ, complement, or scavenger receptors simultaneously with the addition of LPS markedly inhibited the induction of IL-12(p40) mRNA. The phagocytosis of latex microspheres also inhibited IL-12 mRNA induction, but to a much lesser extent. The suppression of cytokine mRNA induction by receptor ligation was specific to IL-12, and not due to a generalized failure of macrophage function, since the induction of IL-10 and TNF-α mRNA were not inhibited by receptor ligation. In fact, IL-10 mRNA levels from BMMφ incubated with LPS and E-IgG were slightly elevated compared to those from BMMφ incubated with LPS alone (Fig. 2 A). Quantitative RT-PCR was performed to determine the degree of reduction of IL-12(p40) mRNA caused by FcγR ligation. Ligation of macrophage FcγR resulted in a 100–200-fold reduction in the amount of IL-12(p40) mRNA induced in response to LPS (Fig. 2 B).


Selective suppression of interleukin-12 induction after macrophage receptor ligation.

Sutterwala FS, Noel GJ, Clynes R, Mosser DM - J. Exp. Med. (1997)

Effect of specific receptor ligation on the LPS- induced expression of cytokine  mRNA. (A) BMMφ from  BALB/c mice were exposed to  LPS (50 ng/ml), or LPS in combination with either E-IgG, E-C3bi,  E-ML-BSA, or latex microspheres. 6 h after the addition of  stimuli, total RNA was isolated  and used to carry out competitive RT-PCR, using primers for  HPRT, TNF-α, IL-10, or the  inducible p40 subunit of IL-12, as  described in Materials and Methods. (B) cDNA generated from  BALB/c BMMφ exposed to  LPS (50 ng/ml) or LPS in combination with E-IgG, were first  normalized for HPRT levels. Constant volumes of normalized cDNAs were then amplified in the presence of decreasing concentrations of competitor  (PQRS), using primers for the inducible p40 subunit of IL-12. The concentration of the experimental cDNA is represented by the equivalent intensities  of competitor and wild-type bands. The fold decrease in IL-12(p40) levels between BMMφ exposed to LPS or LPS in combination with E-IgG can be  determined by taking the ratio of their equivalence points.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196339&req=5

Figure 2: Effect of specific receptor ligation on the LPS- induced expression of cytokine mRNA. (A) BMMφ from BALB/c mice were exposed to LPS (50 ng/ml), or LPS in combination with either E-IgG, E-C3bi, E-ML-BSA, or latex microspheres. 6 h after the addition of stimuli, total RNA was isolated and used to carry out competitive RT-PCR, using primers for HPRT, TNF-α, IL-10, or the inducible p40 subunit of IL-12, as described in Materials and Methods. (B) cDNA generated from BALB/c BMMφ exposed to LPS (50 ng/ml) or LPS in combination with E-IgG, were first normalized for HPRT levels. Constant volumes of normalized cDNAs were then amplified in the presence of decreasing concentrations of competitor (PQRS), using primers for the inducible p40 subunit of IL-12. The concentration of the experimental cDNA is represented by the equivalent intensities of competitor and wild-type bands. The fold decrease in IL-12(p40) levels between BMMφ exposed to LPS or LPS in combination with E-IgG can be determined by taking the ratio of their equivalence points.
Mentions: BMMφ from BALB/c mice were incubated for 6 h with either LPS alone or simultaneously with LPS and particulate ligands (Fig. 2 A). LPS was a potent inducer of IL-12(p40), IL-10, and TNF-α mRNA. The ligation of Fcγ, complement, or scavenger receptors simultaneously with the addition of LPS markedly inhibited the induction of IL-12(p40) mRNA. The phagocytosis of latex microspheres also inhibited IL-12 mRNA induction, but to a much lesser extent. The suppression of cytokine mRNA induction by receptor ligation was specific to IL-12, and not due to a generalized failure of macrophage function, since the induction of IL-10 and TNF-α mRNA were not inhibited by receptor ligation. In fact, IL-10 mRNA levels from BMMφ incubated with LPS and E-IgG were slightly elevated compared to those from BMMφ incubated with LPS alone (Fig. 2 A). Quantitative RT-PCR was performed to determine the degree of reduction of IL-12(p40) mRNA caused by FcγR ligation. Ligation of macrophage FcγR resulted in a 100–200-fold reduction in the amount of IL-12(p40) mRNA induced in response to LPS (Fig. 2 B).

Bottom Line: The results of several different experimental approaches suggest that IL-12 downregulation was due to extracellular calcium influxes that resulted from receptor ligation.Finally, bone marrow-derived macrophages from FcR gamma chain-deficient mice, which fail to flux calcium after receptor ligation, failed to inhibit IL-12(p40) mRNA induction.These results indicate that the calcium influxes that occur as a result of receptor ligation are responsible for inhibiting the induction of IL-12 by LPS.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.

ABSTRACT
Interleukin (IL)-12 is a monocyte- and macrophage-derived cytokine that plays a crucial role in both the innate and the acquired immune response. In this study, we examined the effects that ligating specific macrophage receptors had on the induction of IL-12 by lipopolysaccharide (LPS). We report that ligation of the macrophage Fcgamma, complement, or scavenger receptors inhibited the induction of IL-12 by LPS. Both mRNA synthesis and protein secretion were diminished to near-undetectable levels following receptor ligation. Suppression was specific to IL-12 since IL-10 and tumor necrosis factor-alpha (TNF-alpha) production were not inhibited by ligating macrophage receptors. The results of several different experimental approaches suggest that IL-12 downregulation was due to extracellular calcium influxes that resulted from receptor ligation. First, preventing extracellular calcium influxes, by performing the assays in EGTA, abrogated FcgammaR-mediated IL-12(p40) mRNA suppression. Second, exposure of macrophages to the calcium ionophores, ionomycin or A23187, mimicked receptor ligation and inhibited IL-12(p40) mRNA induction by LPS. Finally, bone marrow-derived macrophages from FcR gamma chain-deficient mice, which fail to flux calcium after receptor ligation, failed to inhibit IL-12(p40) mRNA induction. These results indicate that the calcium influxes that occur as a result of receptor ligation are responsible for inhibiting the induction of IL-12 by LPS. Hence, the ligation of phagocytic receptors on macrophages can lead to a dramatic decrease in IL-12 induction. This downregulation may be a way of limiting proinflammatory responses of macrophages to extracellular pathogens, or suppressing the development of cell-mediated immunity to intracellular pathogens.

Show MeSH
Related in: MedlinePlus