Detection of Reactive Oxygen Intermediate Production by Macrophages
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The reparative phenomena that follow sterile tissue injury are an ordered sequence of partially overlapping events and include the macrophage as a key cell type in orchestrating the process of repair. Several studies suggest that the wound-derived macrophage acquires a functional phenotype that is congruent with its specialized role in repair, and that this phenotype is determined by factors within the wound milieu. This phenotype distinguishes the wound macrophages from macrophages obtained at other anatomical sites. Evidence in support of this hypothesis includes the findings that wound-derived macrophages are more phagocytic and fungicidal than resident or immuneelicited peritoneal macrophages (
1
). Wound macrophages demonstrate a substantially greater capacity to suppress splenocyte proliferation in a nitric oxide (NO)—dependent mechanism but fail to kill NO-sensitive tumor cells that readily succumb to immune activated cells (
2
,
3
). Wound macrophages also demonstrate differential responsiveness to activation signals. Exposure of wound macrophages to anoxia results in an increase in arginase activity not seen by similar exposure of immune-elicited or resident peritoneal cells (
4
). Wound macrophages are deficit in their capacity to generate reactive oxygen intermediates (ROIs) even when challenged with classic activators of the respiratory burst oxidase system (Fig. 1 Fig. 2 Fig. 3 4 ) (
5
). Several assays for measuring ROI production in inflammatory cells are provided in this chapter.
Fig. 1.
Evidence that wound-derived macrophage lysates do not demonstrate NADPH oxidase activity as determined by reduction of ferricytochrome-
c
recorded at 550 nm. Arrows indicate the times of addition for PMA (200 n
M
), deoxycholate/Tween (DOC), NADPH (0.6 m
M
), and SOD (300 U/mL). (Reproduced with permission from
ref.
5
).
Fig. 2.
H
2
O
2
release by peritoneal lavage or wound-derived macrophages. H
2
O
2
release after PMA stimulation (200 n
M
) was measured over time using 4-OH phenylacetic acid and HRP. When indicated, SOD (300 U/mL) was present in the cuvets prior to the addition of PMA. Wound-derived macrophages: (−) SOD (■); (+) SOD (●). Peritoneal lavage cells: (−) SOD (□), (+) SOD (◯). (Reproduced with permission from
ref.
5.
)
Fig. 3.
O2
2
−
-dependent lucigenin chemiluminescence. Peritoneal lavage or wound macrophages were dispensed at 10
7
cells/mL into 0.5 mL of HBSS containing 200 μ
M
lucigenin and placed in the chamber of a Chem-Glo II photometer. Cells were stimulated with PMA (200 n
M
), and the chemiluminescent signal was recorded and expressed in volts. The square wave was generated using a radioactive standard and indicates similar instrument calibration for both runs. Chemiluminescence was completely abrogated by SOD (300 U/mL) (not shown). (Reproduced with permission from
ref.
5.
)
