| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 8, 2000 as doi:10.1096/fj.00-0608fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Laboratory of Host Defenses, NIAID, NIH, Bethesda, MD 20892
2Correspondence: Building 10, Room 11N106, National Institutes of Health, Bethesda, MD 20982. E-mail: tleto{at}nih.gov
SPECIFIC AIMS
Our objective was to determine whether the genetic deficiency of p47phox, an essential component of the phagocyte NADPH oxidase (phox), renders murine microglial cells unable to produce superoxide in response to phorbol myristate acetate (PMA) or opsonized zymosan (OZ), which are agonists known to trigger superoxide release from circulating phagocytes (neutrophils, macrophages, monocytes) and microglia. In addition, we examined the effects of retroviral transduction with p47phox cDNA on the production of superoxide by p47phox-deficient microglia as a means of demonstrating the direct involvement of NADPH oxidase in superoxide release by microglia.
PRINCIPAL FINDINGS
1. p47phox is detected in wild-type, but
not in p47phox-deficient
(p47phox ‘knock-out’) microglial cells
Primary microglial cultures derived from wild-type (WT) and
p47phox-deficient (KO) mice were compared for production of
p47phox by indirect immunofluorescence. p47phox
was detected in microglial cells derived from WT mice (Fig. 1A
) but not in cells from KO mice (Fig. 1B
).
The p47phox staining appeared as a diffuse cytoplasmic
pattern with the greatest intensity within perinuclear regions of WT
cells, whereas the KO microglia did not stain positively. Only weak,
nonspecific secondary antibody staining of astrocytes was observed in
KO cultures. These findings were consistent with earlier findings,
which showed that targeted disruption of the p47phox locus
resulted in the absence of detectable p47phox in circulating
neutrophils.
|
2. Recombinant p47phox is detected in
retrovirally transduced microglial cells
To address directly the function of p47phox in
microglial cells, retroviral transduction with a recombinant
p47phox-MFGS expression vector was used to introduce
p47phox into KO microglial cultures. Intense
p47phox-specific immunofluorescence was evident in
transduced WT microglial cells (WT+) (Fig. 1C
) and
indicated over-expression of recombinant p47phox at levels
exceeding endogenous p47phox observed in untransduced WT
cells (Fig 1A
). Immunochemical detection of
p47phox in KO microglial cells that were transduced
retrovirally with recombinant p47phox (KO+) is shown in Fig. 1D
. These micrographs also reveal that astrocytes, which
were detected in these microglial cell-enriched fractions, were
transduced with recombinant p47phox.
3. Microglia from p47phox-deficient
(p47phox "knock-out") mice exhibit a
deficiency in oxidative responses
Because of the established role of p47phox as an
essential component of the phagocyte NADPH oxidase, the presence of
p47phox in WT microglia suggested that this protein also
participates in superoxide production in these cells. Therefore,
microglial cultures derived from WT and KO mice were compared for
oxidative responses to stimulation by PMA or OZ. Essentially no
activity was observed in KO microglial cell cultures after their
stimulation by either agonist (Fig. 2A
); however, both agonists elicited significant
superoxide production in WT microglia (Fig. 2B
).
As observed by other investigators, PMA caused greater production of
superoxide in microglia than that yielded by stimulation with OZ.
|
4. Retroviral transduction with p47phoxcDNA restores superoxide release in
p47phox-deficient microglial cells
We tested the activity of microglial cells that were transduced
with a retrovirus containing the human p47phox cDNA to
address directly the involvement of this protein in these oxidative
responses. Data in Fig. 2
indicate that this procedure significantly
restored (KO+; A) or enhanced (WT+; B)
superoxide production in microglia under OZ- or PMA-stimulated
conditions. In agreement with observations in untransduced WT cells
(Fig. 2B
), PMA was a stronger stimulus of superoxide
production than OZ in these corrected (KO+) microglial cell cultures,
although the levels of correction were still less than that observed in
untransduced WT microglia. These data provide a novel genetic
demonstration that p47phox participates in superoxide
release from cultured murine microglial cells, likely serving as an
essential component of an NADPH oxidase functionally related to the
neutrophil system. The similar kinetics of OZ-induced superoxide
production observed in transduced KO (KO+) (Fig. 2C
)
and untransduced WT (Fig. 2D
) microglial cells provide
further support for the notion that the same enzyme was functioning in
all cases.
CONCLUSIONS
This investigation conclusively shows that p47phox
participates in superoxide anion generation from cultured murine
microglial cells (Fig. 3
). For the first time, direct comparisons between wild-type and
p47phox-deficient microglial cells demonstrated significant
differences in superoxide production in response to PMA or OZ.
Immunocytochemical detection of p47phox only in wild-type
cells suggested that the absence of this protein in
p47phox-deficient microglia was responsible for the
inability of these cells to produce superoxide. Accordingly,
transduction of p47phox-deficient cells with a retrovirus
containing the human p47phox cDNA was sufficient to restore
superoxide release in these cells, and restoration of superoxide
production correlated with detection of recombinant p47phox
in these cells. Furthermore, enhanced expression of p47phox
following transduction of wild-type microglia also resulted in
supernormal production of superoxide. The kinetics of superoxide
production observed following activation with OZ were similar in both
the untransduced wild-type and the genetically reconstituted cultures,
consistent with the same enzyme functioning in each of these cultures.
|
The importance of this study is threefold. First, these findings represent the first non-pharmacological evidence indicating that a ‘phagocyte-like’ NADPH oxidase is operative in microglia and can serve as a source of reactive oxygen species (ROS) in the brain. Second, these experiments demonstrate the function and genetic origin of p47phox in microglial cells. Finally, these data suggest that patients with chronic granulomatous disease may be at reduced risk for oxidative damage in the brain, but could be deficient in an as-yet-unknown physiological manifestation of NADPH oxidase function (i.e., brain development). Additional studies are required to determine whether p47phox interacts with other genuine phox components or with their homologs to produce superoxide in microglia.
The phagocyte enzyme consists of five essential components:
gp91phox, p22phox, p47phox,
p67phox, and Rac 1 (macrophages) or Rac 2
(neutrophils). Both gp91phox and p22phox comprise
membrane-imbedded subunits of flavocytochrome b558, while the latter
three are cytosolic components that assemble with the cytochrome at the
phagolysosomal or plasma membrane upon activation. gp91phox
is regarded as the central, core component of the enzyme, because
electron transfer occurs among co-factors associated with this
polypeptide (FAD and two hemes). p47phox and other cytosolic
components are considered necessary co-factors that regulate the
activity of the flavocytochome but do not by themselves generate ROS.
Thus, the effects of p47phox transduction in the
oxidase-deficient microglia suggest the presence of other essential
oxidase components in these cells. Oxidase activation in microglia by
ß-amyloid, OZ, or PMA, agonists that promote NADPH oxidase activity
in circulating phagocytes, and its potentiation by interferon-
and
tumor necrosis factor-
are other indications that the phagocyte
oxidase may function in microglia. Furthermore, superoxide generation
in sheep microglia was blocked by diphenyleneiodonium, a flavoenzyme
inhibitor and a 91 kDa membrane protein was detected in lysates of
these cells with antibody against gp91phox. Finally, an
immunoreactive homolog of p22phox was detected in these
cells, although this peptide exhibited an apparent molecular mass of
29 kDa. Although these data suggest that an NADPH oxidase produces
superoxide in microglial cells, they remain inconclusive because
iodonium compounds inhibit a variety of other flavoprotein enzymes
(NADH dehydrogenase, NO synthase, and xanthine oxidase) and recognition
of common epitopes does not establish common genetic origins of these
antigens with genuine phox components. Thus, information
regarding the genetic origins of the enzyme that releases superoxide
from microglia is necessary to definitively determine whether an enzyme
identical to the phagocyte NADPH oxidase functions in these cells.
Although it appears that microglial cells share common ontogenic origins with other circulating myeloid cells, such as neutrophils, monocytes, macrophages, and eosinophils, several other oxidases (Mox, Tox, and Renox) related to the phagocyte system have now been recognized. However, these oxidases generally exhibit limited expression patterns that may relate to tissue-specific functions (i.e., in colon, thyroid gland, and kidney). Work is in progress to examine whether microglial cells derived from the gp91phox-deficient mouse produce superoxide anions.
The cell-type that contributes most significantly to ROS production in the brain in response to injury or pro-inflammatory conditions remains unclear, although microglia appear to be early reactants to such events. It is possible that ROS derived from stimulated microglia impose the initial insults, while subsequent ROS production is derived from peripherally circulating neutrophils and monocytes. By performing a series of allografts between wild-type and gp91phox-deficient mice, researchers showed that gp91phox expressed in both brain parenchymal cells and in circulating phagocytes contributes significantly to cerebral ischemia-reperfusion-mediated injury; however, this study did not delineate what resident brain cells were involved. Recent work with cultured gp91phox-deficient sympathetic neurons suggests that a neuronal NADPH oxidase directly contributes to neurodegeneration following nerve growth factor deprivation, but it is not known whether NADPH oxidase exists in cerebral neurons and, if so, whether it becomes activated during cerebral ischemia-reperfusion. The microglial oxidase may be activated following ischemia-reperfusion, because cultured microglia produce enhanced amounts of superoxide following serial exposures to hypoxia and reoxygenation. These results, combined with the findings of this report, suggest that ischemia-reperfusion events in the brain may stimulate the microglial NADPH oxidase to produce ROS.
Regardless of their ontogenic origin, microglia can produce significant amounts of superoxide and nitric oxide. The ability of microglia to liberate ROS has made them suspected contributors to a variety of brain diseases. Increasing evidence, such as the genetic proof provided here, indicates that the NADPH oxidase of microglia is likely a major source of ROS in the brain, particularly in response to injury and inflammatory stimuli.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0608fje To cite this
article, use (December 8, 2000) FASEB J. 10.1096/fj.00-0608fje 
This article has been cited by other articles:
|
|
 |
|
  W.-L. Song, J. A. Lawson, D. Reilly, J. Rokach, C.-T. Chang, B. Giasson, and G. A. FitzGerald Neurofurans, Novel Indices of Oxidant Stress Derived from Docosahexaenoic Acid J. Biol. Chem., January 4, 2008; 283(1): 6 - 16. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Bedard and K.-H. Krause The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology Physiol Rev, January 1, 2007; 87(1): 245 - 313. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ueyama, M. R. Lennartz, Y. Noda, T. Kobayashi, Y. Shirai, K. Rikitake, T. Yamasaki, S. Hayashi, N. Sakai, H. Seguchi, et al. Superoxide Production at Phagosomal Cup/Phagosome through {beta}I Protein Kinase C during Fc{gamma}R-Mediated Phagocytosis in Microglia J. Immunol., October 1, 2004; 173(7): 4582 - 4589. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Pao, E. A. Wiggs, M. M. Anastacio, J. Hyun, E. S. DeCarlo, J. T. Miller, V. L. Anderson, H. L. Malech, J. I. Gallin, and S. M. Holland Cognitive Function in Patients With Chronic Granulomatous Disease: A Preliminary Report Psychosomatics, June 1, 2004; 45(3): 230 - 234. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
