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From the Medical Research Council Centre for Inflammation
Research,*
University of Edinburgh, Edinburgh, United
Kingdom; the La Jolla Institute for Allergy and
Immunology,
University of California, San
Diego, California; and the Department of
Medicine,
University of Cape Town, Cape Town,
South Africa
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) isolated from inflamed glomeruli or
generated by interferon-
and lipopolysaccharide treatment in
vitro induce glomerular mesangial cell apoptosis by hitherto
incompletely understood mechanisms. In this report we demonstrate that
nitric oxide-independent killing of co-cultured mesangial cells by
interferon-/lipopolysaccharide-activated M is suppressed by
binding/ingestion of apoptotic cells and is mediated by tumor necrosis
factor (TNF). Thus, soluble TNF receptor-1 significantly
inhibited induction of mesangial cell apoptosis by 1) rodent M in
the presence of nitric oxide synthase inhibitors or 2) human
M, both situations in which nitric oxide release was
minimal. Furthermore, murine TNF knockout M were completely
unable to induce mesangial cell apoptosis in the presence of nitric
oxide synthase inhibitors. We conclude that TNF-restricted
M-directed apoptosis of glomerular mesangial cells can be
down-regulated by M binding/ingestion of apoptotic cells,
suggesting a new mechanism for negative feedback regulation of M
controls on resident cell number at inflamed sites.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) can direct apoptosis of such cells,4
mesangial cell killing being mediated by nitric oxide (in a rodent cell
system) and another unknown factor. Because inflammatory M can
delete neutrophils5
and a range of tumor
cells6-8
by tumor necrosis factor (TNF)-mediated
mechanisms, there was a strong possibility that activated M might
use TNF to induce mesangial cell apoptosis, particularly in human cell
systems in which M production of nitric oxide is notoriously
difficult to detect.9
Furthermore, the demonstration that
activated/inflammatory M can kill resident glomerular cells
immediately begs the question as to how the killing capacity of M
might be regulated. Importantly, work from Reiter and
colleagues10
demonstrated that the capacity of rodent bone
marrow-derived M stimulated with interferon (IFN)- and
lipopolysaccharide (LPS) to induce tumor cell apoptosis was diminished
to 
30% of control by ingestion of apoptotic cells. However,
although this was associated with a modest reduction in nitric oxide
production to 75% of control, the M killing mechanism(s)
suppressed by ingestion of apoptotic cells was/were not characterized
further.10
In this study we set out to determine whether activated M induction
of glomerular mesangial cell apoptosis was suppressed by M ingestion
of apoptotic cells and to determine which M mechanisms for
triggering apoptosis in neighboring mesangial cells were subject to
such control.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Media and fetal calf serum (FCS) were purchased from Life Technologies (Paisley, UK). Tissue culture plastic was from Falcon (Becton Dickinson, Mountainview, CA) and Costar (Cambridge, MA) as stated in the text. Cytokines were purchased from R&D Systems (Minneapolis, MN) and all other reagents from Sigma (St. Louis, MO) unless otherwise stated.
Cell Isolation and Preparation
Human mesangial cells were obtained from the cortex of fresh
nephrectomy specimens. Mesangial cells were purified from outgrowths of
whole, purified glomeruli and passaged according to standard techniques
in full Dulbecco’s modified Eagle’s medium (DMEM)/F12 (with 10% FCS,
and supplemented with penicillin and streptomycin (Life
Technologies).2
Rat mesangial cells were derived from
outgrowths of whole glomeruli as described.4
They were
passaged according to standard techniques in full DMEM/F12, and were
used between passage 6 and 14. Rodent macrophages were derived from
bone marrow taken from the femur of Wistar rats or from murine strains
(TNF
/ß-/-) and wild-type littermate controls (C57BL6 x
129sv). In the TNFR1-Fc blockade studies, they were all C57BL6). Marrow
was prepared and cultured in Teflon bags with murine M-CSF as
previously described.4
Cells were used after 7 to 10 days,
then were plated into wells 16 hours before experimentation to ensure
adequate adhesion and correct cell number. Human macrophages were
derived from peripheral blood monocytes. Briefly, peripheral blood
mononuclear cells were obtained from the buffy coat of fresh peripheral
blood from healthy donors. Red cells were removed by dextran
sedimentation and granulocytes were separated by centrifugation through
a discontinuous Percoll gradient.11
The monocytes were
then purified using the MACS monocyte cell isolation system (Miltenyi
Biotech, Cologne, Germany). Purity of monocytes (>95%) was
established by flow cytometric characteristics. Monocytes were then
cultured in Teflon wells (2 x 106/ml) in
Iscove’s-modified DMEM with 10% autologous serum. Medium was changed
on day 2 and day 4. Differentiation was confirmed by cytology on
cytospins and CD14 immunofluorescence (not shown).
Generation of Apoptotic Cells
Rat mesangial cells were induced into apoptosis by ultraviolet irradiation. Subconfluent monolayers in T75 flasks (Costar) were exposed to UV irradiation (312 nm, 8 W, 3 minutes) followed by incubation for 16 hours. Nonadherent (apoptotic) cells were removed by agitation and collected in the supernatant. After centrifugation, apoptotic cells were further purified by washing with phosphate-buffered saline (PBS) 1x and centrifugation (190 x g, 5 minutes). Apoptosis was confirmed by histology, selective uptake of Hoechst 33342 (1 µg/ml), but exclusion of propidium iodide (PI) (1 µg/ml). Typically, <10% of cells were PI-positive and <5% did not exclude trypan blue (0.2%).
Mouse thymocytes were prepared from 20 g C57/BL6 mice by pressing the thymus through a 50-µm sieve. The single cell supernatant was resuspended (2 x 106 cells/ml) in RPMI supplemented with glutamine and 2-mercaptoethanol in addition to 10% FCS and antibiotics. Cells were either exposed to a 5-minute burst of UV irradiation (312 nm, 8 W) followed by 2.5 hours of culture, or dexamethasone (1 µmol/L) followed by culture for 6 hours. Typically >50% of induced cells were apoptotic (Annexin V binding; Boehringer Mannheim. Mannheim, Germany) and permeable to Hoescht 33342 (1 µg/ml)) whereas <5% of those were positive for the uptake of PI (flow cytometric analysis, data not shown).
Co-Culture of M and Mesangial Cells
For a detailed description, see our earlier work.4
Matured rodent bone marrow-derived macrophages were plated in 96-well
plates initially at a density to cover 60 to 70% of the well surface;
typically this required 2 x 104cells per
well. Rat mesangial cells were prelabeled with CellTracker Green
5-chloromethylfluorescein diacetate CMFDA (Molecular Probes,
Eugene, OR): cell cultures, 70 to 80% confluent, were washed with
medium lacking serum and then incubated for 1 hour in serum-free medium
containing CMFDA at 5 ng/ml. Cells were washed in medium containing
10% FCS to remove any unbound CMFDA, then trypsinized and added to
cultured rodent M in a 1.5 M:1.0 mesangial cell ratio, previously
shown to be optimal for demonstration of macrophage-directed mesangial
cell apoptosis.4
Experiments were performed in DMEM/F12
medium containing 10% FCS. Once cells had become adherent, typically 2
to 4 hours, wells were washed to remove nonadherent cells. In our
earlier work4
mixing unlabeled and labeled cells showed no
evidence of transfer of CMFDA from labeled to unlabeled cells. Rodent
co-cultures were activated with IFN- (100 U/ml) plus LPS (1
µg/ml).
In some experiments, unlabeled apoptotic cells (1 x
105
per well of a 96-well plate) were added to
the established co-culture and to the control wells of mesangial cells
alone, at the same time as activating cytokines. As a control for
apoptotic cells, aliquots of 10-µm diameter sterile latex beads
(Polysciences, UK) were added to adjacent co-culture. When thymocytes
were used as apoptotic cells, aliquots were added to co-culture for 6
hours, then noningested cells gently washed away, before activation. In
separate wells containing M alone, apoptotic cells or latex beads
were also incubated for 6 hours, then washed off, and wells were fixed
for quantification of percentage M that had phagocytosed apoptotic
cells. This was assessed by phase contrast microscopy. M (34 ±
5.6%) phagocytosed at least one apoptotic thymocyte and 30 ±
6.7% phagocytosed apoptotic mesangial cells. M (67 ± 8.4%)
ingested latex beads. By contrast only 1 ± 1% of M
phagocytosed the population of live thymocytes in this assay.
For human co-culture, human monocyte-derived M were plated at 2
x 104
per well as above. Cycling human mesangial
cells were primed with IFN- (500 U/ml) for 24 hours, then prelabeled
with CMFDA as described for rat mesangial cells. Once washed and
trypsinized, they were added to wells in a 2:1 ratio (because of the
larger size of human mesangial cells). After 4 hours, wells were washed
and replaced with full DMEM/F12. Human co-cultures were activated with
human IFN- (Peprotech) (500 U/ml) and LPS (1 µg/ml).
Assessment of Mesangial Cell Apoptosis in Co-Culture
For a detailed description, see our earlier work.4
At
the end of co-culture experiments, wells were either fixed with
formaldehyde (4% final concentration) and stored for 48 hours at 4°C
to allow firm adhesion of apoptotic cells to the plate, or they were
assessed live by fluorescence microscopy after the addition of Hoechst
33342 (1 µg/ml) and PI (1 µg/ml). For assessment of live cells,
green rounded-up cells were scored as apoptotic if they also were
positive for Hoechst uptake, but excluded PI. For assessment of fixed
(and permeabilized) cells by morphology, plates were first
counterstained with PI (1 µg/ml) and Hoechst (1 µg/ml) in PBS for 5
minutes (which stains both M and mesangial cells). After discarding
the stain, wells were covered with a fluorescent mountant. Apoptotic
mesangial cells were easily discernible by green fluorescence and
characteristic morphology. For both live and fixed wells, apoptosis of
mesangial cells was assessed blindly and five fields per well were
randomly chosen without observer bias. Each experiment was performed in
triplicate. Previous studies have shown this method to give similar
results to flow cytometric assays.4
However, the
microscopical assay is reproducibly more sensitive because apoptotic
mesangial cells tend to disintegrate during centrifugation once they
have been fixed. Note that when exogenous apoptotic cells were added
these were not labeled with fluorescent dyes, enabling confident
identification of apoptosis in the previously healthy target mesangial
cells.
Assays of TNF- and Nitric Oxide
Culture supernatants (free from phenol red) were harvested,
clarified by centrifugation (4000 x g, 5 minutes) then
stored at -20°C. After complete thawing, 50 µl of each sample was
assayed by Quantikine mouse TNF- enzyme-linked immunosorbent assay
(R&D Systems) according to the manufacturer’s instructions. A standard
curve with absorbencies from 0.1 to 1.2 was achieved on each occasion.
Samples (50 µl) were assayed for nitrite by mixing with an equal
volume of the Griess reagent as previously described6
and
measuring absorbency at 540 nm and comparing with a sodium nitrite
standard curve.
Chimeric Soluble Death Receptors
Fusion proteins were constructed using cDNAs for the extracellular domains of human receptors fused with the Fc portion of human IgG1. Proteins were expressed in insect cells infected with recombinant baculoviruses. Protein secreted into the culture supernatant was then purified by protein A-Sepharose column affinity. The protein was stored at -20°C in Hanks’ salt solution.12
Knockout Mice
TNF-/TNF-ß double-knockout mice were generated by inserting a
targeting vector between exon 1 and 2 of the murine TNF-ß gene and
the middle of exon 4 of the adjacent murine TNF- gene of chromosome
17 of GS1 mouse embryonic stem cells. Mutant embryonic stem cells were
selected and injected into C57/BL6 blastocysts.13
Statistics
All experiments were performed on at least four separate occasions using at least four animals. The data were expressed as mean values with the SE of mean. Paired data were compared using the t-test and multiple comparisons using analysis of variance.
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Selectively
Suppresses Nitric Oxide-Independent Induction of Mesangial Cell
Apoptosis
In our previous studies of IFN-/LPS-activated rodent bone
marrow-derived M killing of rodent mesangial cells primed with
IFN- (Figure 1)
we demonstrated that
at least half of the mesangial cell killing was independent of
M-derived nitric oxide, being unaffected by inhibitors of inducible
nitric oxide synthase (iNOS) such as L-NMMA at 500
µmol/L.4
We also observed that the vast majority of
mesangial cells induced into apoptosis by activated M were not
ingested,4
presumably because of kinetic and spatial
restraints in a two-dimensional culture system in which IFN--primed
mesangial cells were added above a sparse monolayer of M before the
co-culture was activated by addition of IFN- and LPS.
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capacity to trigger apoptosis in mesangial cells, an established
co-culture of IFN--primed rat mesangial cells with rat M was
exposed to a fivefold excess of mesangial cells induced into apoptosis
by UV irradiation; the apoptotic cells were administered at the same
time as activation of the co-culture with IFN- and LPS. Under
standard conditions, there was no significant effect of apoptotic cells
on M induction of mesangial cell apoptosis. However, in the presence
of 200 µmol/L L-NMMA, apoptotic mesangial cells exerted a dramatic
inhibitory effect on M induction of mesangial cell apoptosis (Figure 2)
. Addition of the same number (1
x 105
per well) of 10-µm latex beads, as a
control phagocytic particle, had no inhibitory effect whether L-NMMA
was present or not (Figure 3)
.
Furthermore, suppression of nitric oxide-independent triggering of
apoptosis was observed with rodent thymocytes induced into apoptosis by
UV or dexamethasone, but not when freshly isolated nonapoptotic
thymocytes were used as controls (Table 1)
. Finally, similar results were
observed whether apoptotic cells were added simultaneously with
activation of co-culture, as above, or if apoptotic cells were
administered to co-culture before activation (Table 1)
. This indicates
that the suppressive effect was mediated by effects of apoptotic cells
on the M most likely consequent on ingestion of apoptotic cells, but
not excluding an effect of binding alone.
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Exposure of activated monocytes/macrophages to apoptotic cells
exerts a well-established inhibitory effect on secretion of
TNF.14,15
It therefore seemed especially likely that
nitric oxide-independent induction of IFN--primed mesangial cell
apoptosis by activated M, which we had found to be inhibited by
apoptotic cells (Figure 2)
, was also mediated by M-derived TNF.
To seek a role for TNF in M directed killing of mesangial cells, we
examined the effect of soluble chimeric death receptors (fused with the
Fc portion of human IgG1) on activated rat
macrophage killing of primed rat mesangial cells in the presence of
inhibitors of nitric oxide synthesis (in these experiments the
selective iNOS inhibitor L-NIL at 30 µmol/L, as validated in our
earlier work4
). A role for M-derived FasL had already
been ruled out in rodent co-culture4
by using bone marrow
M from gld/gld mice (that lack active FasL) and therefore
soluble inhibitors of Fas-FasL interaction were not used. Induction of
rat mesangial cell apoptosis in activated co-culture was assessed at 24
hours. TNFR1-Fc (10 µg/ml) was able to reduce significantly the
killing capacity of M in co-culture (Figure 4)
whereas lymphotoxin ß-receptor
fusion protein (LTßR-Fc16
) and herpesvirus entry
mediator fusion protein (HVEM-Fc17,18
) had no effect. At
higher concentrations, TNFR1-Fc (50 µg/ml) was able to increase
further the reduction in killing capacity (14.3 ± 3.3% rat
mesangial cell apoptosis in activated co-culture with
IgG1 versus 3.9 ± 0.7%
mesangial cell apoptosis with 50 µg/ml TNFR1-Fc), but the reduction
was not complete. Control rat mesangial cells growing alone with 50
µg/ml TNFR1-Fc showed 1.3 ± 0.7% apoptosis at 24 hours. The
IC50 for this fusion protein was 5.0 ± 1.2
µg/ml (Figure 5)
.
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induction of mesangial cell
apoptosis, because human M production of nitric oxide is notoriously
difficult to elicit.9
Indeed, we confirmed that the
IFN-/LPS activation regimen had no significant stimulatory effect on
human monocyte-derived M production of nitric oxide (data not shown)
and that L-NMMA had no effect on activated human M killing of
IFN--primed human mesangial cells (14.7 ± 2.1% apoptosis at
24 hours with 200 µmol/L L-NMMA versus 15.7 ± 1.9%
under control conditions). In this physiologically nitric
oxide-independent human cell system, soluble TNFR1 again demonstrated
selective inhibition of activated M direction of mesangial cell
apoptosis (Figure 6)
.
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Although the foregoing studies provided strong evidence that
ligation of TNFR1 played a major role in nitric oxide-independent M
killing of mesangial cells, the data did not provide direct evidence
that M-derived ligands for TNFR1 (TNF- and TNF-ß) were
involved. Furthermore, the failure of sTNFR1 to exert complete
inhibition of killing might have reflected its physical exclusion from
regions of close contact between macrophages and mesangial cells. To
examine this question definitively, we prepared bone marrow-derived
(BMD) M from double-knockout Tnf-/-,
Tnfß-/- mice, and wild-type littermate controls; all
animals used were C57/BL6 x 129sv to ensure that strain
differences did not complicate interpretation.13
Preliminary studies showed both knockout and wild-type M produced
similar amounts of nitric oxide in response to IFN- with LPS
(50 ± 4.6 [wild type] versus 47.0 ± 3.3
[Tnf/ß-/-] nmol nitrite per
106
cells per 24 hours) and that this was almost
completely inhibited by the iNOS inhibitor L-NIL at 30 µmol/L (to
3.7 ± 2.1 and 2.9 ± 1.9 nmol nitrite per
106
cells per 24 hours, respectively).
Supernatants from M activated with IFN- and LPS confirmed that
the Tnf/ß-/- M were completely unable to produce
TNF-, as assessed by enzyme-linked immunosorbent assay of culture
supernatants at 24 hours (wild-type M 3982 pg/ml
Tnf/ß-/- M< 0.00 pg/ml). Rat mesangial cells were
primed for 24 hours with IFN- then co-cultured with either wild-type
M or Tnf/ß-/- M. The co-cultures were activated
with IFN- (100 U/ml) and LPS (1 µg/ml) in the presence of L-NIL
(30 µmol/L). In these experiments knockout M were completely
unable to induce mesangial cell apoptosis compared with controls of
mesangial cells growing alone in the presence of cytokines (Figure 7)
. Wild-type M induced mesangial cell
apoptosis as expected. These data demonstrate that when nitric oxide
synthesis was inhibited, and therefore used as a model of human
M-directed apoptosis, M production of TNF-/ß accounted for
all of the killing effect of rodent M on mesangial cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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can kill tumor
cells,6-8
it has only very recently become apparent that
macrophages can also direct tissue remodeling by inducing physiological
cell death, in nontransformed resident cells.4,19,20
Thus
we have shown previously that activated M, whether isolated directly
from experimentally inflamed glomeruli or generated by IFN-/LPS
activation in vitro can induce apoptosis in cultured primary
mesangial cells.4
In this report, we demonstrate for the
first time that activated M induction of apoptosis in nontransformed
cells can be profoundly inhibited by interaction of M with apoptotic
cells. However, in a rodent system this effect could only be
demonstrated when nitric oxide production was inhibited. Nevertheless,
such nitric oxide-independent direction of mesangial cell apoptosis was
inhibitable by soluble TNF receptor in both a rodent cell culture
system and a physiologically nitric oxide-independent human cell
culture system. Indeed, a major role for TNF in the rodent system was
confirmed by the failure of knockout M to induce nitric
oxide-independent mesangial cell apoptosis. Therefore, our key
conclusion is that macrophage-derived TNF may play a major role in
directing apoptosis of primary, nontransformed glomerular mesangial
cells.
We also regard it as interesting and important that TNF-restricted M
direction of mesangial cell apoptosis was selectively suppressed by
interaction of M with apoptotic cells but not when healthy cells or
latex beads were used as control particles. These data are in keeping
with the marked suppressive effects of apoptotic cells on activated
rodent bone marrow-derived M killing of tumor cells, in which the
reported data favor a major role for a nitric oxide-independent cell
killing mechanism because nitric oxide release was only modestly
suppressed.10
Both the latter study and the current work
may have methodological differences from experiments in which nitric
oxide-directed parasite killing was suppressed by M binding of
apoptotic cells.21
Nevertheless, a second key conclusion
of our work is that interaction with apoptotic cells can suppress
TNF-restricted M direction of mesangial cell apoptosis.
We propose that further work should examine the likelihood that
activated M deletion of resident cells at inflamed sites may be
subject to negative feedback control, in which ingestion of apoptotic
cells down-regulates M capacity to induce apoptosis in resident
cells. This new concept suggests additional deleterious consequences
should M clearance of apoptotic cells be defective. Previously it
has been proposed22,23
that reduced M capacity for
clearance of leukocytes and other cells undergoing apoptosis, as now
demonstrated in C1q-/- knockout mice,24
threatens tissue injury because of the likelihood that cellular
contents escaping from noningested apoptotic cells undergoing secondary
necrosis will injure tissues directly and indirectly23
by
stimulating M release of injurious mediators. Our new data indicate
that failure to ingest apoptotic cells might deprive activated M of
a crucial off-signal resulting in undesirably prolonged capacity to
direct death of neighboring cells by TNF-restricted mechanisms.
Nevertheless, it is likely to be some time before this hypothesis can
be tested in vivo, because a growing body of evidence argues
that redundancy in clearance mechanisms may require that a number are
disabled before sustained defects in clearance of apoptotic cells can
be demonstrated.25,26
Furthermore, future work will also need to address whether M
ingestion of apoptotic cells down-regulates M membrane TNF-
cell-surface expression, or whether integrin co-factors, necessary for
successful transduction of the apoptotic signal, are
down-regulated.5
Preliminary data suggest that
membrane-bound rather than soluble TNF- is responsible for
M-directed apoptosis of mesangial cells (J. Duffield and J. Savill,
unpublished data), as reported for M-directed leukocyte
apoptosis.5
To conclude, when macrophage release of nitric oxide is minimal, as may
be expected in human inflammation, these data demonstrate a major role
for TNF in activated M killing of cytokine-primed mesangial cells by
apoptosis. Furthermore, inhibition of the capacity to direct apoptosis
consequent on M binding/ingestion of apoptotic cells reveals a new
and potentially important negative feedback control mechanism in
remodeling of the inflamed site.
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Acknowledgements |
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Footnotes |
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Supported by a Medical Research Council Clinical Training Fellowship (no. G84/4757, to J. S. D.) and a Wellcome Trust Program Grant (no. 047273, to J. S.).
Accepted for publication July 5, 2001.
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