Related Experiment Videos
Granulocyte-activating mediators (GRAM): III. Further functional characterization of monocyte-derived GRAM
A Kapp1, G Zeck-Kapp, A Möller
1Department of Dermatology, University of Freiburg, FRG.
Abstract:
As shown previously monocytes upon stimulation with bacterial lipopolysaccharides (LPS) release granulocyte-activating mediator(s) (M-GRAM) which induced a long-lasting chemiluminescence (CL) response in human granulocytes. M-GRAM could be separated from interleukin-1 alpha and beta, interleukin-2, interferon alpha and gamma, granulocyte colony stimulating factor (G-CSF) and macrophage colony stimulating factor (M-CSF), since these cytokines are shown to be unable to induce a significant CL response. In contrast, granulocyte macrophage colony stimulating factor (GM-CSF) and particularly tumor necrosis factor (TNF) are important triggers of the oxidative burst and they are capable of inducing a CL response. TNF activity but not lymphotoxin (LT) activity could be demonstrated in M-GRAM samples. A polyclonal rabbit IgG as well as a monoclonal antibody to recombinant human TNF which neutralized the TNF activity in M-GRAM preparations did not substantially block the CL signal. Furthermore, M-GRAM-induced CL response was not significantly inhibited by a polyclonal calf antiserum to human recombinant GM-CSF. For further functional characterization of M-GRAM-induced granulocyte activation different assays were performed in order to compare GM-CSF and TNF: (a) SOD-inhibitable cytochrome C-reduction (.O2-); (b) horseradish peroxidase-mediated oxidation of phenol red (H2O2); (c) the release of peroxidase; (d) ultrastructural detection of hydrogen peroxide production; and (e) scanning and transmission electron microscopy (SEM and TEM). Significant release of .O2- was induced by M-GRAM, TNF, and GM-CSF, whereas H2O2 production was significantly stimulated only by M-GRAM and TNF, as shown by functional and ultrastructural assays.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Monocytes release a granulocyte-activating mediator (M-GRAM) that triggers a chemiluminescence response. While tumor necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) contribute, M-GRAM uniquely stimulates hydrogen peroxide production.
Area of Science:
- Immunology
- Cellular Biology
- Biochemistry
Background:
- Monocytes stimulated by lipopolysaccharides (LPS) release granulocyte-activating mediators (M-GRAM).
- M-GRAM induces a chemiluminescence (CL) response in human granulocytes.
- Previous studies identified M-GRAM as distinct from various cytokines like ILs, IFNs, G-CSF, and M-CSF.
Purpose of the Study:
- To functionally characterize M-GRAM-induced granulocyte activation.
- To compare the effects of M-GRAM, TNF, and GM-CSF on granulocyte responses.
- To investigate the role of TNF and GM-CSF in M-GRAM activity.
Main Methods:
- Assays for superoxide anion (.O2-) and hydrogen peroxide (H2O2) production.
- Measurement of peroxidase release.
- Ultrastructural and electron microscopy (SEM, TEM) for H2O2 detection.
- Neutralization assays using antibodies against TNF and GM-CSF.
Main Results:
- M-GRAM, TNF, and GM-CSF induced significant superoxide anion (.O2-) release.
- M-GRAM and TNF significantly stimulated hydrogen peroxide (H2O2) production, unlike GM-CSF.
- Antibodies to TNF and GM-CSF did not fully inhibit M-GRAM-induced CL response, suggesting other components in M-GRAM.
Conclusions:
- M-GRAM is a potent activator of granulocyte oxidative burst, distinct from TNF and GM-CSF in its H2O2-stimulating capacity.
- The M-GRAM complex contains TNF activity but is not solely composed of TNF or GM-CSF.
- Further research is needed to fully elucidate the composition and mechanism of M-GRAM.