Avid Bioservices Inc (CDMO)

[jazzbeerman]

Peter Henson's insightful Oct 2006 PS / TGF-beta paper --------



--------


Apoptotic cells, through transforming growth factor-beta, coordinately induce
anti-inflammatory and suppress pro-inflammatory eicosanoid and NO
synthesis in murine macrophages.



Celio G. Freire-de-Lima∗†‡2, Yi Qun Xiao†‡, Shyra J. Gardai†, Donna L. Bratton†, William P.
Schiemann† and Peter M. Henson†
*Instituto de Biofísica Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro,
RJ 21944-970, Brazil
†Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research
Center, 1400 Jackson Street, Denver, Colorado 80206

Running title: TGF-beta modulates inflammatory eicosanoids



link to full text:
http://www.jbc.org/cgi/reprint/M605146200v1



snips below-

j


--------------------



In the studies reported here, we showed that the
TGF-beta induction by apoptotic cells was
dependent on exposed PS

...............

These results strongly
suggest that the apoptotic cell inhibition of
pro-inflammatory mediator production is
pleiotropic and significantly dependent on the
stimulation of TGF-beta production.

...............

The
implication is that recognition of PS drives the
production of TGF-beta and the downstream antiinflammatory
responses reported herein.

..............

The induction of TGF-beta itself
could be attributed to exposed
phosphatidylserine on the apoptotic cells,
which therefore, appears to drive the
balanced inflammatory mediator responses.

..............

Apoptotic cells are rapidly engulfed by
adjacent tissue cells or macrophages before
they can release pro-inflammatory/proimmunogenic
intracellular contents. In
addition, recognition of the apoptotic cells is
actively anti-inflammatory and antiimmunogenic
with generation of antiinflammatory
mediators such as
transforming growth factor-beta (TGF-beta) and
anti-inflammatory eicosanoids. Here, we have
investigated the role played by the induction
of TGF-beta in the coordinate expression of antiinflammatory
eicosanoids or PPARγ and in
the suppression of pro-inflammatory lipid
mediators and nitric oxide (NO).

................

As a
cell becomes apoptotic, it is generally removed
in situ by near-neighbor cells or macrophages in
a quiet, almost invisible fashion; that is, the
process does not induce a local tissue reaction.
In fact, recognition and removal of apoptotic
cells is normally both anti-inflammatory and
anti-immunogenic (6-9).

................

The interaction and recognition are triggered by
surface changes on the apoptotic cells.

.................

there is considerable evidence to implicate PS as
the main stimulus for the anti-inflammatory or
anti-immunogenic effects (6-8,14-16).

...............

A major anti-inflammatory mediator induced in
response to apoptotic cells, mAb217 or PS
liposomes is TGF-beta (6,8,16). Blockade of TGF-
beta has been shown to reverse the suppressive
effects of apoptotic cells or PS in vivo on either
inflammation or adaptive immunity (7,8).

................

A key issue, therefore, is whether apoptotic cellinduced
TGF-beta, acting in an autocrine/paracrine
fashion, mediates the alterations in eicosanoid
generation. By use of a dominant negative TGF-
beta receptor construct we have been able to show
that apoptotic cells stimulate via their induction
of active TGF-beta, a co-ordinate production of
generally anti-inflammatory, and simultaneous
inhibition of generally pro-inflammatory,
eicosanoids.

................

Results
Apoptotic cells or antibody to PSRS on murine
macrophages stimulate production of TGF- beta
and concomitant blockade of LPS-induced
TNF alpha, NO and iNOS.

.................

Discussion
Apoptotic cells are known to induce an antiinflammatory
and anti-immunogenic response,
in part mediated by their induction of active
TGF-beta in responding cells. Here we show that
the effect of the apoptotic cells is to drive a
complex coordinated inhibition of potentially
inflammatory mediators along with induction of
potentially anti-inflammatory molecules in
macrophages that are orchestrated by the TGF-beta
production.

.................

The observations required the demonstration of
TGF-beta production in response to the apoptotic
cells – shown earlier by numerous investigators
and confirmed herein. A number of ligands have
been demonstrated on apoptotic cells that can
interact with a number of “receptors” on
responding cells, in this case macrophages.
Additionally there are a large group of “bridge”
molecules (see ref. 39) that can link the
apoptotic cell ligands to the receptors. We have
suggested that two important ligands are
phosphatidylserine (PS) and calreticulin. The
latter, as well as the collectin family of bridge
molecules (40) has been suggested to interact
with LRP as a receptor and, in isolation, seems
to induce a more pro-inflammatory response
(5,13). On the other hand, PS and its receptors
and possibly some or all of its bridge molecules
appear to induce the anti-inflammatory effects
and, in most cases, to act in a dominant fashion
in the normal response to apoptotic cells.
Necrotic cells are usually thought to be proinflammatory
(see for example 9,15) and may
have reversed this PS-driven dominance. Other
studies that have suggested that apoptotic cells
can in some circumstances act in a proinflammatory
fashion may also reflect variations
in balance between pro-inflammatory (e.g. LRP)
versus anti-inflammatory (e.g. PS driven)
responses.

................

The results indicate a complex effect of
apoptotic cells acting through release of TGF-beta
to upregulate generally anti-inflammatory
mediators and inhibit the production of proinflammatory
molecules.


________________________________________________



FULL TEXT BELOW -



__________________________________________________



Apoptotic cells, through transforming growth factor-β, coordinately induce
anti-inflammatory and suppress pro-inflammatory eicosanoid and NO
synthesis in murine macrophages.




Apoptotic cells are rapidly engulfed by
adjacent tissue cells or macrophages before
they can release pro-inflammatory/proimmunogenic
intracellular contents. In
addition, recognition of the apoptotic cells is
actively anti-inflammatory and antiimmunogenic
with generation of antiinflammatory
mediators such as
transforming growth factor-β (TGF-β) and
anti-inflammatory eicosanoids. Here, we have
investigated the role played by the induction
of TGF-β in the coordinate expression of antiinflammatory
eicosanoids or PPARγ and in
the suppression of pro-inflammatory lipid
mediators and nitric oxide (NO). By use of a
dominant negative TGFβII receptor, TGF-β
signaling was blocked and its participation in
the consequences of apoptotic cell stimulation
determined. The induction of TGF-β itself
could be attributed to exposed
phosphatidylserine on the apoptotic cells,
which therefore, appears to drive the
balanced inflammatory mediator responses.
Arachidonic acid release, COX-2 and
prostaglandin (PG) synthase expression were
shown to be significantly dependent on the
TGF-β production. On the other hand, a
requirement for TGF-β was also shown in the
inhibition of thromboxane synthase and
thromboxanes, of 5-lipoxygenase and
sulfidopeptide leukotrienes as well as iNOS
and NO. TGF-β-dependant induction of
arginase was also found and would further
limit the NO generation. Finally, apoptotic
cells stimulated production of 15-
lipoxygenase and 15-HETE, a potentially
anti-inflammatory pathway acting though
PPARγ, and lipoxin A4 production which
were also upregulated by a TGF-β-dependant
pathway in this system. These results strongly
suggest that the apoptotic cell inhibition of
pro-inflammatory mediator production is
pleiotropic and significantly dependent on the
stimulation of TGF-β production.



Recognition and clearance of apoptotic cells by
phagocytes play pivotal roles in development,
maintenance of tissue homeostasis, control of
the immune response, and resolution of
inflammation (1,2). Apoptotic cells are removed
by professional phagocytes, members of the
mononuclear phagocyte system (MPS) such as
macrophages and immature dendritic cells, or by
non-professional phagocytes such as fibroblasts,
endothelial, epithelial, smooth muscle or stromal
cells (3). Uptake of the apoptotic cell is by a
specialized and highly conserved form of
phagocytosis termed efferocytosis (4,5). As a
cell becomes apoptotic, it is generally removed
in situ by near-neighbor cells or macrophages in
a quiet, almost invisible fashion; that is, the
process does not induce a local tissue reaction.
In fact, recognition and removal of apoptotic
cells is normally both anti-inflammatory and
anti-immunogenic (6-9).
The interaction and recognition are triggered by
surface changes on the apoptotic cells. Two
widely distributed surface ligands on apoptotic
cells are phosphatidylserine, PS (10,11) and
calreticulin (5) which become associated in
patches together on the cell surface. Indirect
effects of the collectin family of molecules or
direct action of calreticulin leads to stimulation
of LRP (LDL receptor related protein) on the
phagocytosing cell (5,12). However, LRP
activation seems to induce production of proinflammatory
mediators (13). On the other hand,
there is considerable evidence to implicate PS as
the main stimulus for the anti-inflammatory or
anti-immunogenic effects (6-8,14-16). We
suspect that these two stimuli, acting through
different signaling pathways, are balanced, with
a normal bias towards the anti-inflammatory.
Unfortunately, the receptor(s) that recognizes PS
(PS recognition structures, PSRS) that is
responsible for this effect is unknown although it
does seem to distinguish between stereoisomeric
forms of the phosphoserine head group (10,14)
and does seem to react with an activating IgM
antibody mAb217 (17). whose binding is
blocked by PS. The antibody binds to, and
activates cells and mimics exactly the effects of
PS on apoptotic cells in contributing to uptake
and on the generation of anti-inflammatory
mediators (7,18) and has been used here along
with apoptotic cells to stimulate macrophages
for production or suppression of eicosanoids.
A major anti-inflammatory mediator induced in
response to apoptotic cells, mAb217 or PS
liposomes is TGF-β (6,8,16). Blockade of TGF-
β has been shown to reverse the suppressive
effects of apoptotic cells or PS in vivo on either
inflammation or adaptive immunity (7,8). On the
other hand, earlier studies also suggested
induction of other candidates such as IL-10 (19),
PGE2 (6) and even PAF (6) although the last
two can have both pro- or anti-inflammatory
effects. The ability of apoptotic cell recognition
to alter the production of eicosanoids had first
been noted for thromboxane (20,21) and
exemplified by roles for TGF-β and
prostaglandins in apoptotic cell enhancement of
Trypanosoma cruzi growth in macrophages (22).
A key issue, therefore, is whether apoptotic cellinduced
TGF-β, acting in an autocrine/paracrine
fashion, mediates the alterations in eicosanoid
generation. By use of a dominant negative TGF-
β receptor construct we have been able to show
that apoptotic cells stimulate via their induction
of active TGF-β, a co-ordinate production of
generally anti-inflammatory, and simultaneous
inhibition of generally pro-inflammatory,
eicosanoids. The effect is mediated by effects on
the synthases for these mediators. Additional
coordinate effects were seen on related proteins
including iNOS which was downregulated and
PPARγ or arginase, which were induced, i.e.
combining to reduce NO production and also
potentially in keeping with the antiinflammatory
balance.



Results
Apoptotic cells or antibody to PSRS on murine
macrophages stimulate production of TGF- β
and concomitant blockade of LPS-induced
TNF α, NO and iNOS.
Murine macrophages (peritoneal or RAW 264,
1.0 x106 cells/ml) were stimulated with LPS
(100 ng/ml) as positive control or mAb217 (50
µg/ml) or apoptotic Jurkat T cells (3x106
cells/ml) for 18 h. These stimuli each induced
TGF-β production in both types of macrophages
(Figure 1A). Isotype control IgM or viable
Jurkat T cells were inactive. These data are in
accordance with previous reports (6,16). The
stimulation of TGF-β production by apoptotic
cells in this system was blocked by
preincubation of the targets with the PS-binding
protein Factor Va (Figure 1B) as had been
shown earlier with annexin V (29). PS
liposomes also stimulated the production of
TGF-β but less efficiently. This is probably
because the presentation to the PSRS from PS
exposed on the apoptotic cell is from a quite
different environment compared with a
liposome. However, PS liposomes increased the
production of TGF-β in the presence of LPS or
Cyclosporine A (Figure 1C). These findings
suggest that PS liposomes themselves may upregulate
TGF-β translation when TGF-β
message has been induced by other stimuli
(17,30). Classically activated macrophages (LPS
and IFNγ stimulation) exhibit release of TNFα
and NO as well as upregulation of iNOS. As
shown in figures 1D-E, these three responses to
stimulation with LPS and IFNγ were inhibited
by exposure of the macrophages to apoptotic
cells or mAb217. Previous studies implicated the
TGF-β produced, in the suppression of TNFα
induction and might be expected to serve the
same role for suppression of iNOS and NO.
To demonstrate this presumed requirement for
TGF-β in the suppression, a dominant negative
form of the TGFβ RII was employed.
Transfection of RAW 264 cells with this
construct was shown to block the ability of
TGF-β to signal for 3 x PT-luc reporter (which
contains three consecutive TPA response
elements (TREs) and a portion of the
plasminogen activator inhibitor (PAI-1)
promoter region) gene activation (Figure 2A).
Since TGF-β can induce its own synthesis, the
effect of the dominant negative receptor was
also examined on the production of TGF-β itself
after stimulation with apoptotic cells, mAb217
or LPS. As shown in Figure 2B, this treatment
blocked 60-70% of the TGF-β produced by each
of the stimuli i.e. supporting an additional
autocrine/paracrine effect of TGF-β on its own
induction in these systems.
Transfection of the truncated TGFβRII was
found to completely reverse the suppression of
TNFα and NO production caused by apoptotic
cells or PSRS stimulation and also restored the
upregulation of iNOS protein (Figures 2C-E). In
keeping with the suppression of NO production
by blocking upregulation of iNOS, exposure of
macrophages to apoptotic cells or PSRS stimuli
also led to increases in intracellular levels of
arginase 1 (Figure 2F) which could further
reduce the production of nitric oxide.
Macrophages with truncated TGF βRII are
defective in prostaglandin production and
prostaglandin synthase expression in response
to stimulation with apoptotic cells or LPS.
In the original studies of anti-inflammatory
effects of apoptotic cells, PGE2 was also shown
to be generated and it too seemed to play a role
in suppression of inflammatory mediators (27).
Accordingly we next examined the effect of
apoptotic cells and stimulation with mAb217 on
induction of potentially anti-inflammatory
prostaglandins as well as the role of TGF-β in
their regulation. The original studies did not
address the probable induction of PGI2 (detected
as PGF1α) along with PGE2 and, accordingly,
this was included in the analysis. Supernatants
from the cell culture were collected 18 h after
stimulation and analyzed for PGE2 and PGF1α.
The cell lysates were collected and the levels of
synthases for PGE2 (PGES1), PGD2 and PGI2
determined by Western blotting. As expected,
the two stimuli induced production of PGE2 and
PGF1α starting at 2 h or earlier and extending
out to 18 h incubation (Figure 3A-C).
Importantly, they also increased the intracellular
levels of the prostaglandin synthases (Figure
3D).
It has previously been reported that TGF-β can
induce prostaglandin production (22,31,32) and
in data not shown, direct addition of active TGF-
β to the macrophage cultures did stimulate
production of PGE2 and PGF1α. When the
macrophage response to TGF-β was blocked
with the dominant negative receptor, induction
of PGE2 and PGF1α by either apoptotic cells or
mAb217 was prevented (Figure 3A and B).
Interestingly, LPS-induced PGE2 and PGF1α
were also reduced by about 80%. We suspect
that the lack of complete blockade with this
stimulus reflects the possible use of alternative
pathways not involving TGF-β. In keeping with
the data on the prostaglandins themselves, the
truncated receptor also reduced upregulation of
the synthases (Figure 3D) although not to as
great an extent as seen for the secreted
prostaglandins.
These prostaglandins are lipid mediators that
like TGF-β have been reported to have pro- or
anti-inflammatory properties in different
circumstances. For example, we earlier showed
PGE2 to decrease TNF-α production from
macrophages (6). A possible contributory effect
of prostaglandins themselves to T G F-β
production is depicted in Figure 3E wherein
indomethacin was shown able to reduce the
amount of TGF-β produced in response to
apoptotic cells and mAb217 (see also ref 6).
This, along with the autostimulation of TGF-β
by TGF-β (see above), further shows the
extensive feedback responses inherent in these
systems.


TGF- β-dependent suppression of thromboxane
synthesis.
Early studies of responses to apoptotic cells
showed that thromboxane production was
decreased (20,21) and also see ref. (6). This
suggests co-ordinate up-regulation of potentially
anti-inflammatory prostanoids along with downregulation
of pro-inflammatory thromboxane
and raises the question of whether TGF-β is
responsible for both effects. Since macrophages
do not produce TXA2 spontaneously, and did
not do so at any time after incubation with
apoptotic cells or mAb217 alone (data not
shown), they were activated with LPS in order to
demonstrate the suppressive effect of costimulation
with either apoptotic cells or
mAb217 (Figure 4A). Almost complete
inhibition of TXA2 (measured as TXB2) was
seen, and transfection of the macrophages with
the dominant negative TGF-β receptor reversed
this inhibition. Since LPS also induces TGF-β
production, enhancement of the LPS effect on
thromboxane production might have been seen if
this TGF-β was blocked. However, this was not
observed, probably because the generation of
active TGF-β following LPS occurred after the
majority of the thromboxane had already been
produced. Once again, the effect of the TGF-β
appeared to be at the level of the synthase.
Thromboxane synthase levels in the
macrophages were suppressed by apoptotic cells
and mAb217, but not after transfection of the
truncated TGF-β receptor (Figure 4B).
Stimulation of macrophages with mAb217 or
apoptotic cells induced COX-2 expression and
arachidonic acid release through TGF- β
dependant signaling
The demonstrated reciprocal effect of TGF-β on
the prostanoid synthases raised the possibility
that the induction of PGE2 and PGI2 reflected a
diversion of the precursor PGH2 from utilization
by thromboxane synthase. However, since the
prostaglandins were increased directly in
macrophages that did not express the
thromboxane synthase, it seemed likely that the
apoptotic cells also led to increased levels of one
or other PGH synthases (COX enzymes) and
thereby, increased production of PGH2. No
evidence was found for altered amounts of
COX-1 (data not shown) but both apoptotic cells
and mAb217, as well as LPS as expected, did
increase the intracellular amounts of COX-2
(Figure 5A). In keeping with the theme of this
study, macrophages expressing the truncated
TGF-β receptor did not show upregulation of
COX-2 in response to the apoptotic cells and
markedly reduced that stimulated by LPS.
In order to initiate eicosanoid production, a
source of free arachidonate must be available
and, presumably, must be initiated by the
apoptotic cell or mAb217 stimulus. This is
shown in Figure 5B where free 3H arachidonate
(including its metabolites) was measured in the
supernatant after previous incorporation into
macrophage phospholipids before stimulation. In
this case, blocking the TGF-β effects with the
dominant negative receptor reduced the amounts
of arachidonate released by about 50% in the
case of all three stimuli. This presumably
reflects either a timing issue as noted for the
LPS-induced thromboxane or the availability of
preformed phospholipases that were stimulated
by either the LPS or PSRS engagement in
addition to an effect from TGF-β. TGF-β has
been reported to induce prostaglandin
production (22,31,32), which would necessitate
its ability to initiate released arachidonic acid, an
effect which was shown here in Figure 5B.
Acting through TGF- β, apoptotic cells or
mAb217 decreased LPS-enhanced 5-
lipoxygenase and leukotrienes but increased 15-
LO, 15-HETE and LXA4 as well as PPAR γ.
The pattern of TGF-β induction of potentially
anti-inflammatory, but suppression of proinflammatory,
eicosanoids was further explored
by examining the effects on lipoxygenases and
leukotrienes. Our earlier study (6) had shown
suppression of leukotriene release from
macrophages by apoptotic cells. Stimulation of
macrophages with apoptotic cells or mAb217 by
themselves did not induce cysteinyl leukotrienes
at 18 h and suppressed that induced by priming
with LPS (Figure 6A). However, a time course
study (Figure 6B) did show an early production
at 2 h but none subsequently. Similarly, LTB4
was seen only 2 h after stimulation, after this
time the production was downregulated (data not
shown). Examination of the key upstream
enzyme, 5-lipoxygenase revealed, as expected,
that its levels were increased after priming with
LPS but that this did not occur in the presence of
the apoptotic cells or mAb217 (Figure 6C). In
the presence of the truncated TGF-β receptor,
the LPS-primed increase in levels of 5-LO was
no longer prevented (Figure 6C). We attribute
the early induction of leukotrienes to an effect of
endogenous 5-LO acting on early released
arachidonate before the TGF-β effect has time to
kick in.
By contrast, when 15-LO was examined, the
apoptotic cell stimulus directly increased
intracellular levels of the enzyme and production
of 15-HETE (Figure 6 D-F). The 15-HETE
production was seen 4-8 h after stimulation and
peaked at 18 h (Figure 6F). Once again, the
induction by apoptotic cells or mAb217 was
blocked in the presence of the truncated TGF-β
receptor. On the other hand, TGF-β did not
appear to play a significant role in the induction
of 15-LO or 15-HETE by LPS. Products of 15-
LO have been suggested to participate in lipoxin
A4 (LXA4) production and activation of PPARγ
(33,34), which may also have anti-inflammatory
effects in macrophages (35-37). Accordingly, we
also examined the effect of apoptotic cells and
mAb217 to induce LXA4 and alter the levels of
PPARγ. As shown in Figure 7A-B, both stimuli
initiated LXA4 production and increased the
amounts of PPARγ protein in the cells, as did
direct addition of TGF-β. This supports a
possible additional anti-inflammatory effect of
apoptotic cells via LXA4 and/or activation of
PPARγ



Discussion

Apoptotic cells are known to induce an antiinflammatory
and anti-immunogenic response,
in part mediated by their induction of active
TGF-β in responding cells. Here we show that
the effect of the apoptotic cells is to drive a
complex coordinated inhibition of potentially
inflammatory mediators along with induction of
potentially anti-inflammatory molecules in
macrophages that are orchestrated by the TGF-β
production. By study of the responses induced
by apoptotic cells in macrophages that are
unresponsive to TGF-β by virtue of their
transfection with a dominant negative TGF-β
receptor, the role of this important mediator on a
wide variety of eicosanoids, nitric oxide and
related molecules was delineated. Thus, the
earlier demonstration that apoptotic cells
induced the production of PGE2 but suppressed
thromboxane (6,20) were shown both to be due
to the effects of TGF-β and these observations
extended to other potentially pro- and antiinflammatory
arachidonate metabolites. It
should be noted that the prostanoids PGE2 PGI2
and PGD2 are known to exhibit both pro- and
anti-inflammatory actions, in part for PGE2,
depending upon the receptors that are engaged
(eg (38)). Similar pleomorphic effects should be
noted for NO. In our earlier study (6), we
showed that PGE2 suppressed LPS-induced
inflammatory mediators from macrophages (6)
and in the data reported herein, prostanoid
production also showed a feedback enhancement
of TGFβ production. Thus, in this limited
context we see these prostanoids as potentially
anti-inflammatory and their induction as
contributing to the overall anti-inflammatory
consequences of apoptotic cell recognition.
The observations required the demonstration of
TGF-β production in response to the apoptotic
cells – shown earlier by numerous investigators
and confirmed herein. A number of ligands have
been demonstrated on apoptotic cells that can
interact with a number of “receptors” on
responding cells, in this case macrophages.
Additionally there are a large group of “bridge”
molecules (see ref. 39) that can link the
apoptotic cell ligands to the receptors. We have
suggested that two important ligands are
phosphatidylserine (PS) and calreticulin. The
latter, as well as the collectin family of bridge
molecules (40) has been suggested to interact
with LRP as a receptor and, in isolation, seems
to induce a more pro-inflammatory response
(5,13). On the other hand, PS and its receptors
and possibly some or all of its bridge molecules
appear to induce the anti-inflammatory effects
and, in most cases, to act in a dominant fashion
in the normal response to apoptotic cells.
Necrotic cells are usually thought to be proinflammatory
(see for example 9,15) and may
have reversed this PS-driven dominance. Other
studies that have suggested that apoptotic cells
can in some circumstances act in a proinflammatory
fashion may also reflect variations
in balance between pro-inflammatory (e.g. LRP)
versus anti-inflammatory (e.g. PS driven)
responses.
In the studies reported here, we showed that the
TGF-β induction by apoptotic cells was
dependent on exposed PS by blockade with the
PS-binding protein Factor Va, which has the
advantage over the more usually employed
Annexin V by binding in physiologic
concentrations of calcium. Although direct
stimulation of TGF-β by PS-containing
liposomes was weak, these agents readily
enhanced TGF-β production to other stimuli.
The amounts of TGF-β measured by ELISA
only represents that in the supernatant and is
therefore relatively insensitive for a molecule
that binds to surfaces and cell membranes. Early
in vivo demonstration of PS liposome effects
were also relatively weak in comparison with
apoptotic cells (8) and, we suspect, may reflect
issues of presentation and avidity.
The other approach employed here was to
examine the effect of mAb217, an IgM
activating antibody that is suspected of binding
to an as yet unidentified receptor for PS on
responding cells. Its ability to bind all cells that
respond to PS-exposing apoptotic cells, to be
blocked itself by pretreatment of the cells with
L- but not D-phosphatidylserine, and its close
mimicking of the activation induced by PSexposing
apoptotic cells supports this
contention. In all cases in this study, mAb217
and apoptotic cells behaved identically. The
implication is that recognition of PS drives the
production of TGF-β and the downstream antiinflammatory
responses reported herein.
The eicosanoids are derived from metabolism of
arachidonate through a number of pathways. In
this study we focused on prostanoid synthesis
through PGH synthases (COX) and downstream
prostaglandin and thromboxane synthases as
well as through lipoxygenases and their
downstream products of leukotrienes and
HETES. Apoptotic cell and TGF-β induction of
PGE2, and PGI2 were shown. The effect
appeared to be on upregulation of the respective
synthases PGES and PGIS whose protein levels
were increased in response to the apoptotic cells
and blocked in the presence of the dn-TGF-β
receptor. Likewise, PGD synthase protein levels
were upregulated by the apoptotic cells and
mAb217 but not in the absence of TGF-β
effects. By contrast the pro-inflammatory
eicosanoid thromboxane was suppressed. In this
case resting macrophages or those stimulated
with apoptotic cells alone did not produce
thromboxane or exhibit significant levels of its
synthase. When stimulated to induce
thromboxane synthase by LPS or LPS and IFNγ
however, the apoptotic cells or mAb217
suppressed the upregulation and the mediator
production, again in a TGF-β-dependent fashion.
These effects on prostanoid balance implied an
upregulation of both COX and of sources of
substrate, namely arachidonic acid and both
increases in COX2 (but not COX1) and of
released arachidonate were demonstrated in
response to apoptotic cells. The effect on COX2
was also shown to be dependent on TGF-β (see
41,42) and at least part of the arachidonate
release. TGF-β itself also induced liberation of
arachidonate. While not explored directly herein,
it seems reasonable to assume that the apoptotic
cells and/or TGF-β induce activation of existing
phospholipases 2 and that the TGF-β may also
initiate some upregulation of these that
contributes to greater and/or more prolonged
release of the arachidonate.
Our earlier study had suggested that apoptotic
cells also suppressed the production of
potentially pro-inflammatory sulfidopeptide
leukotrienes (6). Here we show that levels of the
upstream enzyme 5-lipoxygenase, as well as of
the leukotrienes themselves, were also
suppressed by the apoptotic cells and that this
too was reversed in the absence of TGF-β
signaling. The decrease in 5-lipoxygenase levels
noted here are at odds with studies showing
enhanced production in response to TGF-β
during macrophage maturation under the
influence of Vitamin D3 (43). We suspect,
therefore, a discordant effect of TGF-β on
maturing versus mature macrophages.
Intriguingly, and in keeping with the coordinated
anti-inflammatory effects, 15-lipoxygenase was
upregulated and its stable product 15-HETE
increased by the apoptotic cells or mAb217,
again due to TGF-β. Products of 15-HPETE are
generally thought to be anti-inflammatory (44-
47) and some may possibly achieve this effect
via activation of PPARγ (34). Accordingly we
also addressed the effect of the apoptotic cells
and TGF-β pathways on LXA4 production and
the upregulation of PPARγ and showed that
those too were increased.
The other pathway and mediator addressed in
this study was the potentially immunoregulatory
and broad spectrum signaling molecule nitric
oxide (NO). Apoptotic cells and mAb217
suppressed the generation of NO and the
upregulation of iNOS. TGF-β is well known to
inhibit production of iNOS (48-50) and,
therefore, the TGF-β dependence of this was
expected and shown by the dominant receptor
approach. Intriguingly, arginase, which reduces
the substrate for NOS enzymes, was reciprocally
upregulated by the apoptotic cells – again via
TGF-β. This would provide an additional brake
on the NO generation by macrophages
responding to apoptotic cells.
The results indicate a complex effect of
apoptotic cells acting through release of TGF-β
to upregulate generally anti-inflammatory
mediators and inhibit the production of proinflammatory
molecules. The systems employed
here support an effect on synthesis of the various
enzymes involved in generating these mediators,
i.e. acting on transcription, translation or both.
However, we cannot exclude additional effects
on metabolism or secretion of the mediators
acting in the shorter term. In response to
apoptotic cells, macrophages appear to release
preformed TGF-β almost immediately (30-60
minutes, (8,16)) followed by new synthesis and
more prolonged generation of the active
molecule. Combined, this would achieve a fairly
rapid and then prolonged effect on the
inflammatory state of the cell. However, it
should be noted that the immediate generation of
free arachidonate in response to apoptotic cells
might lead to generation of potentially proinflammatory
eicosanoids by enzymatic
pathways constitutively present in the cell (e.g.
5-LO and leukotriene synthases) before the
slower effects of TGF-β had a chance to kick in
(see for example refs. 51-54). Given the
pluripotentiality and plasticity of this cell type,
we are generally skeptical of the concept of
stable macrophage “phenotypes” (3).
Nevertheless, it should also be noted that some
of the effects of apoptotic cell exposure mimic
those seen in the so called alternatively activated
macrophage (55,56) raising intriguing questions
for the future of how long these effects persist
following the interaction with apoptotic cells
and/or the consequences of prolonged exposure
as might be experienced in a resolving
inflammatory response in vivo.



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