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Analysis of the nonfunctional respiratory burst in murine Kupffer cells
1Department of Hematology-Oncology, Cornell University Medical College, New York, New York 10021.
Abstract:
Murine Kupffer cells (KCs), which constitute one of the largest populations of tissue macrophages, differ from most other cells of the myelomonocytic lineage in lacking the capacity for a respiratory burst. A collagenase perfusion technique followed by adherence to plastic at low temperature yielded pure cultures of KCs uniformly expressing receptors for Fc and C3bi, and containing virtually no morphologically detectable intracytoplasmic debris. Such KCs took up and oxidized glucose via the hexose monophosphate shunt about the same as peritoneal macrophages (PCs). Respiratory burst stimuli failed to enhance the hexose monophosphate shunt in KCs, probably because no H2O2 was produced. Detergent-permeabilized KCs generated no O2- in the presence of 1 mM NADPH, in striking contrast to all PC populations studied. Yet, KCs contained at least one component of the O2(-)-producing oxidase, cytochrome b559, in the same quantities as PCs and neutrophils. Cytochrome b559 was demonstrated by a novel double-reduction spectral technique that eliminated interference from hemoglobin and mitochondrial cytochromes. Consistent with the presence of the oxidase, KCs acquired normal respiratory burst capacity after prolonged incubation in vitro. The defect in triggering the respiratory burst in KCs was selective for the reduction of O2 by NADPH, in that reduction of O2 by endogenous arachidonate was readily demonstrate in response to zymosan. The percent of arachidonate released, the percent oxygenated, and the suppression of prostacyclin and leukotriene C production, as well as the pattern of LFA-1 expression, all resembled the pattern reported with PCs several days after exposure to bacteria. Indeed, exposure of PCs to low numbers of zymosan particles led gradually to complete suppression of respiratory burst capacity and refractoriness to its enhancement by rIFN-gamma, as evident in KCs both before and after their explanation. Thus, the modulation of oxidative metabolism that characterizes KCs probably arises from frequent endocytic encounters. This phenomenon may permit macrophages to act as scavengers without oxidative damage to bystander cells.
Insights
Murine Kupffer cells (KCs) lack respiratory burst capacity due to a defect in NADPH oxidase activation, unlike peritoneal macrophages (PCs). This oxidative metabolism modulation may prevent bystander cell damage during scavenging.
Area of Science:
- Immunology
- Cell Biology
- Macrophage Biology
Background:
- Kupffer cells (KCs) are tissue macrophages in mice, known for lacking respiratory burst capacity.
- This deficiency distinguishes them from other myelomonocytic lineage cells.
Purpose of the Study:
- To investigate the underlying mechanisms of the defective respiratory burst in KCs.
- To compare the oxidative metabolism of KCs with peritoneal macrophages (PCs).
Main Methods:
- Isolated pure KC cultures using collagenase perfusion and adherence at low temperature.
- Assessed glucose uptake via the hexose monophosphate shunt.
- Measured superoxide anion (O2-) generation in detergent-permeabilized cells with NADPH.
- Quantified cytochrome b559 using a novel spectral technique.
- Evaluated respiratory burst capacity after in vitro incubation and in response to zymosan.
Main Results:
- KCs showed normal glucose metabolism but failed to enhance the hexose monophosphate shunt with respiratory burst stimuli.
- Permeabilized KCs did not generate O2- with NADPH, unlike PCs.
- KCs possessed cytochrome b559, a component of the O2(-)-producing oxidase.
- In vitro incubation restored normal respiratory burst capacity in KCs.
- The defect was selective for NADPH-dependent O2 reduction; arachidonate-dependent O2 reduction occurred with zymosan.
- KC oxidative metabolism resembled PCs after prolonged bacterial exposure.
Conclusions:
- The defective respiratory burst in KCs is likely due to impaired triggering of NADPH oxidase, not a lack of components.
- Frequent endocytic encounters may modulate KC oxidative metabolism, enabling scavenging without harming bystander cells.