| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Section of Nephrology, Department of Medicine, University of Chicago, Chicago, IL 60637
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
) from several SLE-prone strains
(5, 6, 7, 8, 9).
We have previously shown that peritoneal and bone marrow-derived M
from prediseased MRL/+ and MRL/lpr SLE-prone mice display a
substantial defect in LPS-induced expression of several cytokines,
including IL-1
, IL-1ß, and IL-6 (5, 6, 7, 8, 9, 10, 11). As shown by
the construction of irradiation chimeras in which bone marrow from
SLE-prone MRL mice was transferred into lethally irradiated normal
recipients, and vice versa, this defect is intrinsic to the M and
independent of the host autoimmune environment (8).
Moreover, it is fully manifest in M from mice as young as 1 wk of
age, and neither the pattern nor the magnitude of the defect changes
with age or the development of disease (8).
Underexpression of IL-1 exists at both an mRNA and protein level. It is
transcriptionally determined, and equally affects secreted and
cell-associated IL-1 bioactivity (5, 6, 7, 8, 9, 10, 11). As assessed by
bioassay and densitometry of Western or Northern blots, the defect is
progressive over time, increasing in magnitude from 
2-fold within
the first 24 h of culture to as much as 50-fold by 24–48 h
(5, 6, 7, 8, 9, 10, 11).
To this point, our standard protocol has been to culture M in medium
containing FBS. In the present study, we show that this defect is
eliminated by culture in the absence of FBS. The responsible factor(s)
in FBS includes lipids, as delipidation of FBS fully eliminates the
defect. Moreover, only anionic lipids, as found on the surface of
apoptotic cells, or apoptotic cells themselves can reproduce the
defect. We show further that these results extend identically to
prediseased mice of all other murine models of SLE. Importantly, none
of 13 non-SLE-prone strains possesses a similar defect. Given the
central role of the M in apoptotic cell clearance and Ag
presentation, a defect in the expression of multiple M-derived
cytokines that is triggered by apoptotic cells could have broad
potential to alter the balance between self-tolerance and immunity.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
AKR/J, B6.C3H.gld (B6/gld), B6.MRL.lpr (B6/lpr), BALB/c, BXSB, C3HeJ, C3H/HeN, C57BL/6, (C57BL/6 x New Zealand Black (NZB)) F1, CBA, LG/J, MRL/+, MRL/lpr, NZB, New Zealand White (NZW), NZB/W F1, and SWR/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Unless otherwise indicated, these mice were used between 4 and 6 wk of age. Diseased MRL/lpr and NZB/W F1 mice were retired female breeders, greater than 8 mo of age (The Jackson Laboratory). All animal protocols were approved by the Institutional Animal Care and Use Committee at Boston Medical Center (Boston, MA).
Reagents
Recombinant human plate-derived growth factor-BB (PDGF-BB) and
recombinant human insulin-like growth factor-1 (IGF-1) were obtained
from QCB (Hopkinton, MA). LPS (Escherichia coli derived,
serotype 0111:B4) was obtained from List Biological (Campbell, CA).
Dioleoyl phosphatidylserine (PS), dioleoyl phosphatidylcholine, and
dioleoyl phosphatidylethanolamine (PE) were obtained from Avanti
(Alabaster, AL). Murine TNF- was obtained from Biosource
International (Camarillo, CA). Lysophosphatidic acid (LPA),
lipoteichoic acid (LTA), okadaic acid, zymosan, linoleic acid, and
oleic acid were obtained from Sigma (St. Louis, MO). Latex beads (1.2
µm) were obtained from Seradyne (Indianapolis, IN). LPS-binding
protein (LPS-BP) was the generous gift of Dr. Peter S. Tobias (Scripps
Research Institute, La Jolla, CA). Neutralizing anti-murine IL-10
mAb and neutralizing anti-murine TGF-ß mAb (active against
TGF-ß1, TGF-ß2, and TGF-ß3) were obtained from R&D Systems
(Minneapolis, MN).
M culture
Peritoneal exudate cells were harvested by lavage 3 days after
i.p. injection of 1.5 ml of 4.05% thioglycolate broth
(5, 6, 7, 8). Cells were washed twice in RPMI 1640 and plated in
60 x 15-mm tissue culture dishes at 4 x
106 cells/dish in R.10 culture medium (RPMI 1640
plus 10% FBS, with 2 mM L-glutamine, 5 mM HEPES, 100 U/ml
penicillin, and 100 µg/ml streptomycin). After a 4-h incubation at
37°C, nonadherent cells were removed by washing with RPMI 1640. The
remaining adherent cells, >98% M as determined by morphologic
examination and nonspecific esterase staining, were cultured in R.10 or
FBS-free R.0 medium (R.10 minus FBS) plus human rM-CSF (generous gift
of Genetics Institute, Cambridge, MA). M were used after overnight
culture in their respective medium.
FBS delipidation
FBS was delipidated by one of two protocols. FBS was mixed 1:1 (v/v) for 2 h at 22°C with either butanol/diethyl ether (1:4, v/v) (12) or chloroform/methanol (2:1, v/v) (13), then centrifuged to separate organic and aqueous phases. Residual organic solvents were removed from the delipidated aqueous phase by applying a water-aspirator vacuum for 2 h. The resultant delipidated FBS was sterilely filtered for later use.
Induction of apoptosis
Apoptosis was induced in freshly isolated murine thymocytes by
incubation for 4 h with 5 x 10-6 M
hydrocortisone in either R.10 or R.0 medium (1). Before
addition to M, thymocytes were washed three times in RPMI 1640 and
resuspended in R.0 medium. Viable thymocytes were defined as propidium
iodide (PI)-negative cells with faint nuclear Hoechst staining.
Apoptotic thymocytes were defined as PI-negative cells with bright
nuclear Hoechst staining and decreased cell size. Postapoptotic
thymocytes (i.e., apoptotic thymocytes that had lost cell membrane
integrity) were defined as PI-positive cells with bright Hoechst
staining and decreased cell size. By these criteria, 
50% of
thymocytes were apoptotic, 25% were viable, and 25% were
postapoptotic. The kinetics of induction of apoptosis and the
population distribution at 4 h were identical for thymocytes
incubated in R.10 vs R.0 medium. Necrotic cells, as defined by
increased cell size in association with uptake of PI and faint Hoechst
staining, comprised <0.1% of the final cell population.
Northern blotting
Total RNA was isolated from M using Trizol Reagent (Life
Technologies, Grand Island, NY), according to manufacturer’s
instructions. Equivalent amounts of RNA (usually 10–20 µg) were
applied to 1.2% agarose gels containing 0.66 M formaldehyde, and
subsequently transferred to Nytran membranes (Schleicher & Schuell,
Keene, NH). The RNA blots were then prehybridized, hybridized, and
washed, as recommended by the manufacturer. The IL-1 probe used in
these experiments was a 1.7-kb EcoRI insert fragment from a
plasmid containing the murine IL-1 cDNA. The IL-1ß probe was a
1.4-kB EcoRI insert fragment from a plasmid containing the
murine IL-1ß cDNA. Both plasmids were kindly provided by Dr. Patrick
Gray, Genentech (South San Francisco, CA). The ß-actin probe was a
1.5-kb PstI insert fragment from a plasmid containing the
rat brain ß-actin cDNA (kindly provided by Dr. Steve Farmer, Boston
University School of Medicine, Boston, MA). Insert fragments were
radiolabeled by the random primer method using a kit from New England
Nuclear Research Products (Boston, MA), according to manufacturer’s
specifications. Equal loading of lanes was confirmed by examination of
18S and 28S ribosomal RNA bands, as well as by probing for the
housekeeping gene ß-actin. In all cases, the densitometric difference
among lanes was 
1.5x.
RNase protection assays
RNase protection assays were performed using a RiboQuant In Vitro Transcription Kit (PharMingen, San Diego, CA), according to manufacturer’s instructions. All probes were for murine cytokines.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
from SLE-prone MRL/+ mice is
dependent upon the presence of FBS
Peritoneal M from prediseased SLE-prone MRL/+ mice showed a
profound defect in LPS-induced expression of IL-1 and ß (Fig. 1
, A, B, and
E). This difference in IL-1 expression between nonautoimmune
BALB/c and MRL/+ M increased with time. In the case of IL-1 (Fig. 1, A and E), expression by MRL/+ M at 8 h
was 50% of that by BALB/c M. Although BALB/c M maintained
expression of IL-1 through 24 and 48 h (32% and 6% of that at
8 h), expression of IL-1 by MRL/+ M was virtually
undetectable at these times (<0.5% of that at 8 h). The results
for IL-1ß followed a similar pattern (Fig. 1, A and
B), with expression of IL-1ß by MRL/+ M at 8 h
being nearly equivalent to that by BALB/c M. However, while BALB/c
M maintained expression of IL-1ß at 24 and 48 h (84% and
27% of that at 8 h), expression of IL-1ß by MRL/+ M was
virtually undetectable at these times (<0.5% of that at 8 h).
This defect is not restricted to peritoneal M, but occurs also in
bone marrow-derived M induced to differentiate in vitro by M-CSF
(5, 8). Moreover, the defect is independent of the manner
of M elicitation, being present in thioglycolate-elicited, proteose
peptone-elicited, and resident nonelicited peritoneal M (Refs.
5 and 8 , and not shown).
|
only rarely revealed detectable
IL-1 message at 48 h, even upon prolonged exposure of Northern
blots. To highlight the magnitude of this defect, we have chosen blots
whose exposure emphasizes the striking lack of IL-1 message at the 24-
and 48-h time points (Fig. 1
A). A similar principle guided
our choice of blots in all later figures.
Strikingly, the removal of FBS from the culture medium (FBS-free)
resulted in equalization of IL-1 mRNA expression between MRL/+ and
nonautoimmune BALB/c M (Fig. 1, C, D, and
F). In the absence of FBS, expression of both IL-1 and
ß by MRL/+ M was virtually identical with that of BALB/c M at
all time points (Fig. 1, C and F). Thus, this
MRL/+ defect is distinct from that of the LPS-resistant C3H/HeJ strain,
which carries a mutation in the Tlr4 LPS receptor (14) and
showed impaired induction of IL-1 both in the presence and in the
absence of FBS (Fig. 1, A and D). The inhibitory
effect of FBS on IL-1 expression by MRL/+ M was dose dependent (Fig. 1G).
As FBS-free culture of M required the addition of M-CSF to maintain
M viability, we confirmed that addition of M-CSF was not responsible
for correcting the defect. Supplementation of FBS-containing medium
with M-CSF had no effect on the defect (Fig. 1H). Moreover,
M-CSF alone, in the absence of LPS, did not induce IL-1 (not shown).
Another potential contributory factor in FBS-free culture is the
absence of LPS-BP, which enhances the interaction of LPS with the CD14
receptor (15, 16). Consistent with its role solely as a
catalyst, the addition of LPS-BP to FBS-free medium did not
reconstitute the IL-1 defect (Fig. 1H). Together, these data
indicate that MRL/+ M are able to respond normally to LPS, but that
an unidentified factor(s) in FBS results in inhibition of LPS-induced
IL-1 expression. It should be noted that FBS-free culture also
increased the magnitude of IL-1 expression by BALB/c M, although the
degree of change was far less than that seen for MRL/+ M. Thus, the
response of MRL/+ M to FBS may represent an exaggeration of a normal
regulatory response.
FBS-dependent IL-1 underexpression is present in M from all the
major murine models of SLE
FBS-dependent IL-1 underexpression also occurred in M from
prediseased mice of all the other major inbred models of murine SLE
(17) (Fig. 2A).
Importantly, none of six non-SLE-prone strains tested in this study
(AKR/J, BALB/c, C3H/HeN, C57BL/6, CBA/J, and SWR) (Fig. 2, B
and C) or seven strains evaluated previously (A/J, A.Thy,
B.10, B.10A, B.10BR, C3HeB/FeJ, and DBA.2) (5, 6, 7, 8, 9, 10, 11) showed
a similar defect. As opposed to control strains, SLE-prone strains are
characterized by the virtual absence of detectable message at 24 and
48 h, even upon prolonged exposure of Northern blots. This
property of SLE-prone M is represented graphically in Fig. 2, E and F, in which the relative expression at
48 h compared with that at 8 h is shown for all strains under
both FBS-containing (Fig. 2E) and FBS-free (Fig. 2F) conditions. Under FBS-containing conditions, relative
expression at 48 h was 44.8 ± 9.9% for nonautoimmune
strains and 1.7 ± 2% for SLE-prone strains
(p < 0.001). In marked contrast, when M
were cultured in the absence of FBS, there was no difference in
expression at 48 h between nonautoimmune and SLE-prone strains
(79.7 ± 8.6% vs 79.9 ± 5%, p >
0.95).
|
. This was done
to minimize any effects of disease, thereby enabling us to distinguish
those abnormalities that play a fundamental role in the etiology of SLE
from those that are a consequence of established disease. Our results
therefore suggest that FBS-dependent IL-1 underexpression is
independent of disease and may represent a background trait of
SLE-prone mice. Several additional features of this defect bear upon
this point. First, among SLE-prone strains, despite broad variation in
severity and manifestations of disease (17), the pattern
and magnitude of the defect were essentially the same. Second, within
individual strains, the presence of the defect was unaffected by
disease and its inflammatory sequelae. Thus, FBS-dependent IL-1
underexpression was equally manifest in M from both diseased and
prediseased MRL/lpr and NZB/W F1 mice
(Fig. 2A). Finally, as previously shown (8),
IL-1 underexpression is one of the very earliest immunologic
abnormalities detectable in SLE-prone mice, being fully manifest in
MRL/lpr mice as young as 1 wk of age. Intriguingly, LG/J mice, which account for 75% of the genome of MRL mice (17) and themselves manifest systemic autoimmune disease (18), possessed a similar defect. In contrast, the other three nonautoimmune MRL parental strains, which contribute only 25% of the genome (17), showed normal IL-1 expression.
The fact that IL-1 underexpression is indistinguishable between MRL/+
and MRL/lpr mice implies that the genetic basis for this
defect resides in the MRL background and is independent of such genes
as lpr or gld, which exacerbate and accelerate
the course of autoimmunity, but are themselves weak inducers of
autoimmunity (19, 20). This was confirmed by the absence
of a defect in C57BL/6 mice homozygous for either lpr or
gld (Fig. 2D). FBS-dependent IL-1 underexpression
was also independent of the Y-chromosome-linked accelerant gene
Yaa (17, 21), as male and female SLE-prone BXSB
mice had similar defects (Fig. 2A). Finally, no defect was
seen in the nonautoimmune first generation offspring of C57BL/6 and NZB
mice (22) (Fig. 2D). This suggests that at
least one of the genes contributing to this defect is recessive or,
alternatively, that the defect exhibits gene dosage dependency.
Together, these results suggest that FBS-dependent IL-1 underexpression
represents a shared phenotype of murine SLE.
FBS-dependent IL-1 underexpression by MRL M is attributable to
an abnormality(ies) within only one of several signaling pathways
involved in IL-1 induction
FBS-dependent IL-1 underexpression was not limited to induction by
LPS, but also occurred in response to stimulation by other danger
signals, including toxic shock syndrome toxin-1, LTA, and TNF- (Fig. 3A). In contrast, induction of
IL-1 by MRL/+ M in response to the phosphatase inhibitor okadaic
acid or to yeast cell-derived zymosan A particles was identical with
that by BALB/c M despite the presence of FBS (Fig. 3B).
Importantly, induction of IL-1 by LPS vs okadaic acid (23)
or zymosan A (24) occurs by distinctly different
intracellular signaling pathways. These data suggest that the MRL M
defect is attributable to an abnormality within only one of several
signaling pathways involved in IL-1 induction.
|
We next sought to identify the specific FBS-dependent factor(s)
that elicits this defect. Initial characterization of FBS indicated the
necessity of lipid factor(s), as delipidation of FBS by two different
methods (12, 13) restored IL-1 expression by MRL/+ M to
nearly normal levels (Fig. 4A). Consistent with the
requirement of a lipid factor(s), addition to FBS-free medium of
PDGF-BB or IGF-1, two prominent cytokines in FBS, did not lead to a
defect (Fig. 4B). We next attempted to reconstitute the IL-1
defect by addition of various lipid factors to FBS-free medium. Neutral
lipids, such as PE (Fig. 4B) and dioleoyl
phosphatidylcholine (not shown), did not reproduce the defect. In
contrast, the addition to FBS-free medium of several anionic lipids,
including LPA, linoleic acid, and PS, resulted in IL-1 underexpression
by MRL/+ M (Fig. 4B). Although striking, reproduction of
the defect by anionic lipids was variable and inconsistent, suggesting
that they were incomplete stimuli.
|
from all the major
SLE-prone strains
Because apoptotic, but not viable, cells express the anionic lipid
PS on their outer cell membrane (25), we considered the
possibility that apoptotic cells might be a more complete stimulus for
eliciting the defect (Fig. 5A). Apoptotic thymocytes
alone failed to elicit a defect. However, when thymocytes were induced
to undergo apoptosis in the presence of FBS and then extensively
washed, they now yielded a significant defect by MRL/+ M.
Importantly, while neither delipidated FBS (dFBS) nor unopsonized
apoptotic thymocytes alone could reproduce the defect, dFBS-opsonized
apoptotic thymocytes yielded a powerful defect. This result indicates
that interaction of a nonlipid FBS factor, presumably a protein, with
the surface of apoptotic cells creates a ligand capable of eliciting
the defect. As a final control, viable thymocytes (alone or washed
after preincubation with FBS) were unable to produce a defect. Because
viable thymocytes eventually undergo apoptosis during prolonged
culture, this last result suggests that the necessary nonlipid factor
in FBS interacts specifically with the surface of apoptotic, but not
viable, thymocytes and, therefore, is lost during the washing of viable
cells. Although we have previously shown that the abundant FBS protein
ß2-glycoprotein I (ß2GPI) selectively interacts with apoptotic
cells (1), ß2GPI-opsonized apoptotic thymocytes failed
to elicit a defect (not shown). This implies that proteins other than,
or in addition to, ß2GPI may be necessary. The effect of
dFBS-opsonized apoptotic cells on IL-1 expression by MRL/+ M was
dose dependent over a range of apoptotic cell to M ratios extending
from 1:1 to 10:1 (Fig. 5C).
|
.
Latex particles were precultured in either FBS-free medium or 10% FBS
and then extensively washed in a manner identical with that used for
apoptotic thymocytes. Phagocytic uptake of latex particles by MRL/+
M did not elicit a defect in IL-1 expression (Fig. 5B).
To this point, M had been exposed only to syngeneic apoptotic
thymocytes. We next sought to ensure that the defect was intrinsic to
the MRL/+ M, and not the result of a difference between BALB/c and
MRL/+ thymocytes. We therefore exposed MRL/+ and BALB/c M to
syngeneic or heterogeneic FBS-opsonized apoptotic thymocytes (Fig. 5C). Irrespective of the source of thymocytes, BALB/c M
displayed no defect in IL-1 expression, whereas MRL/+ M showed a
dose-dependent defect. This result confirms that the defect resides
within the MRL/+ M.
Consistent with the idea that FBS-dependent IL-1 underexpression
represents a shared phenotype of murine SLE, dFBS-opsonized apoptotic
thymocytes mimicked the effect of FBS and elicited a dose-dependent
defect in IL-1 expression by M from all the major murine inbred SLE
models, as well as the MRL-parental autoimmune LG/J strain (Fig. 6). IL-1 expression by M from five
other representative nonautoimmune strains (AKR/J, C3H/HeN, C57BL/6,
CBA/J, and SWR) resembled that of BALB/c M and was not
down-regulated in response to dFBS-opsonized apoptotic thymocytes (not
shown). Thus, there was full concordance between FBS alone and
dFBS-opsonized apoptotic cells in producing the defect. Furthermore,
delipidation of FBS normalized IL-1 expression in all these same
strains (Fig. 6).
|
extend to multiple other
cytokines in addition to IL-1
We next used an RNase protection assay to determine whether
M-derived cytokines other than IL-1 show a similar FBS-dependent
defect in LPS-induced expression (Fig. 7). As compared with BALB/c M,
FBS-dependent cytokine expression by MRL/+ M followed one of three
patterns: decreased duration and magnitude of expression (IL-1ß,
IL-6, IL-12 p35, IL-12 p40, GM-CSF, M-inflammatory protein-1ß,
RANTES, TNF-); increased expression (IL-10); and equivalent
expression (M-CSF, M migration inhibition factor, TGF-ß1,
TGF-ß2, TGF-ß3). Northern analysis showed that expression of
IL-1 is also decreased (Fig. 1). In all cases, FBS-free culture led
to equalization of LPS-induced expression between MRL/+ and BALB/c
M. As previously noted in the cases of IL-1 and IL-ß (Fig. 1, A and D), FBS-free culture also increased the
magnitude of cytokine expression by BALB/c M, although the magnitude
of change was far less than that seen for MRL/+ M (Fig. 7). Whereas
BALB/c M expressed most cytokines at 24 and 48 h under both
FBS-free and FBS-containing conditions, MRL/+ M expressed these same
cytokines at 48 h only under FBS-free conditions. These data
suggest that the response of MRL/+ M to FBS may represent an
exaggeration of a normal regulatory response.
|
Finally, recent reports have shown that phagocytosis of apoptotic
cells by human M inhibits LPS-induced expression of several
cytokines, including IL-1 (26, 27). Inhibition
appears to be mediated by autocrine release of anti-inflammatory
factors such as IL-10 or TGF-ß1. Because the inhibitory
effect of FBS and dFBS-opsonized apoptotic cells on cytokine production
by MRL/+ M resembles that induced by IL-10 or TGF-ß1, we
determined whether IL-1 underexpression by SLE-prone M could be
attributable to exaggerated release of inhibitory autocrine factor(s).
We addressed this question through three independent sets of studies
(Fig. 8).
|
cultured in the presence of FBS. As shown in Fig. 8A, addition of these neutralizing mAb failed to correct
IL-1 underexpression by MRL/+ M. Although these data definitively
rule out a significant role for IL-10 and TGF-ß in mediating the
defect, they do not rule out a role for other potential autocrine
inhibitory factors such as platelet activating factor or
PGE2 (26).
We therefore designed the following studies using conditioned media
(CM). Our reasoning was as follows. First, if an increase in secreted
anti-inflammatory mediators by SLE-prone M is responsible for IL-1
underexpression at 24 and 48 h, then culturing nonautoimmune M
in CM derived from SLE-prone M stimulated with LPS for 48 h
should result in sharply decreased levels of IL-1 production by
nonautoimmune M. Correspondingly, culturing SLE-prone M in CM
derived from nonautoimmune M should result in increased levels of
IL-1 production by SLE-prone M. Second, if IL-1 underexpression by
SLE-prone M at 24 or 48 h is the result solely of heightened
sensitivity to otherwise normal levels of secreted autocrine mediators,
then culturing SLE-prone M in CM derived from nonautoimmune M
stimulated with LPS for 48 h should result in comparably decreased
levels of IL-1 production by the SLE-prone M.
We generated CM by two means. In the first, BALB/c and MRL/+ M were
cultured in the presence of FBS and stimulated with LPS for 48 h.
MRL/+ CM generated in this manner had no effect on BALB/c IL-1
production (Fig. 8B). Similarly, BALB/c CM had no effect on
MRL/+ IL-1 production (Fig. 8B). Analogous results were
obtained comparing CM from BALB/c vs NZW M (not shown). To eliminate
any potential confounding effect of FBS in CM on IL-1 expression, CM
was also generated by exposure to dFBS-opsonized apoptotic thymocytes.
BALB/c and MRL/+ M were cultured in FBS-free medium and exposed to
dFBS-opsonized apoptotic thymocytes, then stimulated with LPS for
48 h, as done in generating Figs. 5 and 6. Before use, CM was
subjected to ultracentrifugation at 13,000 x g for 30 min
so as to remove all residual apoptotic cells and bodies. BALB/c and
MRL/+ CM generated in this manner also lacked a differential effect on
IL-1 expression by freshly cultured M (Fig. 8C).
Taken together, these results indicate that FBS-dependent IL-1
underexpression is unlikely to represent an exaggeration of the
anti-inflammatory effect of apoptotic cell uptake previously described
in normal M. These results also indicate that IL-1 underexpression
cannot be attributed to a heightened sensitivity of SLE-prone M to
an otherwise normal pattern of secreted autocrine factors. Rather, IL-1
underexpression appears to be the consequence of an
abnormality within a specific signaling pathway that is directly
modulated by serum lipids and/or apoptotic cells.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
from prediseased mice from all the major
murine models of SLE have a defect in cytokine expression that is
triggered by FBS and/or apoptotic cells. Affected strains include
MRL/+, MRL/lpr, NZB, NZW, NZB/W F1,
and BXSB, all of which express or contribute to the expression of
autoimmunity (17). In addition, the MRL parental strain
LG/J, which has recently been shown to develop autoimmunity
(18), also manifests the defect. No similar defect can be
found in 13 nonautoimmune strains, including the remaining three
nonautoimmune MRL parental strains. In addition, the defect is
independent of genes such as lpr, gld, and
Yaa, which accelerate and exacerbate the development of
autoimmunity, but in and of themselves are weak inducers of
autoimmunity.
Elicitation of this defect in M exposed to FBS or apoptotic cells
leads to the dysregulated expression of multiple cytokines. Of 15
cytokines examined, the expression of 9 was down-regulated (IL-1,
IL-1ß, IL-6, IL-12 p35, IL-12 p40, GM-CSF, M-inflammatory
protein-1ß, RANTES, TNF-), the expression of one was up-regulated
(IL-10), and the expression of the remaining 5 was unaffected (M-CSF,
M migration inhibition factor, TGF-ß1, TGF-ß2, TGF-ß3). In all
cases, culture under FBS-free conditions led to equivalent expression
of cytokines. Such broad dysregulation of the pattern of cytokines
produced by M has the potential to perturb the overall cytokine
network and upset the balance among regulatory and effector B and T
cell subsets.
Accumulating evidence suggests that apoptotic cells and their products are the target of autoantibodies across a broad spectrum of autoimmune diseases (1, 2, 3, 4). Many of the most prominent autoantigens in SLE have been shown to localize on the surface of apoptotic cells. These autoantigens include nucleosomal DNA-histone complexes, small nuclear ribonucleoproteins (including the Smith and U1-A Ag), cytoplasmic ribonucleoproteins (SS-A/Ro and SS-A/La), and the target of anti-phospholipid autoantibodies (1, 2, 3, 4). Importantly, self Ag expressed on the surface of apoptotic cells are conformationally intact, as autoantibodies from patients with SLE and/or the anti-phospholipid syndrome are able to recognize and bind to these self Ag on the surface of apoptotic cells. Indeed, the paradigm that autoantigens are expressed on the surface of apoptotic cells may extend to autoimmune diseases other than SLE, as we have shown that myeloperoxidase and proteinase 3, two targets of anti-neutrophil cytoplasmic autoantibodies, are expressed on the surface of apoptotic neutrophils (4).
In addition to surface expression of conformationally intact self Ag,
apoptotic cells may also provide an abundant source of self Ag in the
form of processed peptide fragments that can be recognized by T cells
in the context of MHC molecules. Each day, billions of cells throughout
the body die by apoptosis. A major proportion of these apoptotic cells
is cleared by professional phagocytes, including the M. At least in
the case of MHC class I, peptides from phagocytosed apoptotic cells
have been shown to end up in the peptide groove of MHC molecules
(28, 29).
Thus, apoptotic cells provide a more or less continuous source of self
Ag in two distinct forms, each recognizable by one or the other of the
two major Ag receptors of the immune system. When present as intact
molecules on the surface of apoptotic cells, apoptotic self Ag will be
recognizable by Ab in the form of secreted Ig or membrane-bound Ig
within the B cell Ag receptor. In contrast, when presented as linear
peptide fragments in the context of MHC molecules by APC such as the
M, apoptotic self Ag will be recognizable by TCRs.
The central challenge for an immune system is the discrimination of self from nonself. Although central tolerance mechanisms within the thymus and bone marrow provide an efficient first line of defense against autoimmunity, a certain percentage of potentially autoreactive T and B cells regularly escape deletion and emerge into the periphery. Such escape is most likely a consequence of the close similarity between many foreign and self Ag, and reflects the fact that elimination of all lymphocytes whose receptors bear any affinity for self Ag would leave dangerous holes in the immune repertoire. These potentially autoreactive T and B cells require ongoing monitoring and containment through peripheral tolerance mechanisms (30, 31, 32, 33). We have suggested that presentation of apoptotic Ag may play an active role in maintaining peripheral self-tolerance and that defects in the handling of apoptotic cells have the potential to predispose to autoimmunity (1, 2, 4). It is an axiom of discrimination theory that correct classification, as for example between self and nonself, is considerably enhanced when a template exists for comparison. We propose that apoptotic Ag fulfills the role of template for the immune system, and acts as a renewable, continuously updated source of self Ag against which the immune system can check and reeducate itself.
It is instructive to list the requirements of an ideal template and to examine how well apoptotic self Ag meets these requirements. First, the template should be present wherever needed. Because apoptosis occurs in all organs and tissues throughout the body, apoptotic self Ag exists everywhere that the immune system performs its surveillance. Second, the template should be plentiful. With billions of cells dying each day by apoptosis, apoptotic self Ag should always exist in surplus. Third, the template must be properly tagged as template so it will not be confused with prospective Ag. Although the precise ligands on apoptotic cells that are recognized by phagocytes remain to be identified (34, 35), there are a number of surface changes, such as redistribution of the anionic phospholipid PS from the cytoplasmic to the external leaflet of the cell membrane followed by capture of circulating ß2GPI, that appear to definitely mark cells as apoptotic (1, 23, 36, 37, 38). In addition, as a corollary to the "danger model" (39, 40), apoptotic cell death and clearance occur in the complete absence of inflammation, so there are no danger signals available to induce costimulatory molecules necessary for immune activation (26, 27). Fourth, the template must be easily read by the immune system. As discussed above, apoptotic self Ag not only exists in a conformationally intact form on the surface of apoptotic cells (1, 2, 3, 4), but is also processed by APC and presented as linear peptide fragments in the context of MHC (28, 29). Finally, a mechanism must exist for updating the self-template. In other words, reading of apoptotic self Ag should induce signaling cascades that continually update and alert the immune system to potential changes in the definition of self, as for example may occur during pregnancy or puberty (39, 40).
In terms of this model, one may envision multiple abnormalities that
could predispose an individual to autoimmunity. Examples include the
following: 1) errors in the generation of self-template, perhaps due to
abnormalities in the executionary phase of apoptosis; 2) errors in the
tagging and/or recognition of apoptotic cells as self-template,
involving either the ligands specific to apoptotic cells or the
receptors for apoptotic cells on phagocytes; 3) errors in the
presentation of self-template to the immune system, perhaps due to
abnormalities in processing or presentation of self-Ag by those
phagocytes that also function as APC; and 4) errors in the reading,
interpretation, and/or updating of self-template in response to
apoptotic self Ag. A M abnormality triggered by apoptotic cells,
such as we describe, would act to disrupt peripheral tolerance through
either of the last two mechanisms. This model would also explain the
emergence over time of autoimmunity, as errors accumulated within the
template and eventually exceeded a certain critical threshold.
In summary, FBS- and apoptotic cell-dependent IL-1 underexpression
appears to represent a shared phenotype for murine SLE. This phenotype
may even represent a more generalized feature of autoimmunity, as a
similar defect exists in M from nonobese diabetic mice
(41), which spontaneously develop autoimmune diabetes
mellitus, but not in M from nonautoimmune diabetic strains (J.
S. Koh and J. S. Levine, manuscript in preparation). We
hypothesize that the basis for this defect lies within a specific
signaling pathway that is triggered by the recognition and/or uptake of
apoptotic cells. Although the identity of the responsible lipid
components in FBS remains undetermined, the most likely candidates are
oxidatively modified lipids, lipoproteins, or lipid-protein adducts,
whose uptake by M relies upon many of the same receptors as do
apoptotic cells (42, 43, 44). Although we have focused on
cytokine expression, FBS-dependent abnormalities may affect a broader
range of M functions. For example, in the presence of FBS, MRL M
showed increased adhesion to a variety of extracellular matrix
proteins, whereas, in the absence of FBS, adhesion of MRL M was
identical with that of control M (J. S. Koh and J. S.
Levine, manuscript in preparation). The presence of a shared phenotype
triggered by FBS and/or apoptotic cells among all the major murine SLE
models raises the possibility of a common genetic defect (or separate
defects within a common signaling pathway) linking these SLE-prone
strains.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Jerrold S. Levine, University of Chicago, Section of Nephrology, 5841 South Maryland Avenue, MC 5100, S-506, Chicago, IL 60637.
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; ß2GPI, ß2 glycoprotein I; CM, conditioned medium; dFBS, delipidated FBS; IGF-1, insulin-like growth factor-1; LPA, lysophosphatidic acid; LPS-BP, LPS-binding protein; LTA, lipoteichoic acid; M, macrophage; NZB, New Zealand Black; NZW, New Zealand White; PDGF, platelet-derived growth factor; PE, dioleoyl phosphatidylethanolamine; PI, propidium iodide; PS, dioleoyl phosphatidylserine; R.0, serum-free RPMI-based medium; R.10, RPMI-based medium supplemented with 10% FBS.
Received for publication February 4, 2000. Accepted for publication July 19, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
agonists inhibit production of monocyte inflammatory cytokines. Nature 391:82.
B into human macrophages: lipopolysaccharide-induced, but not zymosan-induced, proinflammatory cytokines are inhibited, but IL-10 is nuclear factor-B independent. J. Immunol. 162:2939.This article has been cited by other articles:
|
|
 |
|
  K Anam, M Amare, S Naik, K. Szabo, and T. Davis Severe tissue trauma triggers the autoimmune state systemic lupus erythematosus in the MRL/++ lupus-prone mouse Lupus, April 1, 2009; 18(4): 318 - 331. [Abstract] [PDF]
|
|
|
|
|
|
S Muller, J Dieker, A Tincani, and P. Meroni Pathogenic anti-nucleosome antibodies Lupus, May 1, 2008; 17(5): 431 - 436. [Abstract] [PDF]
|
|
|
|
 |
|
 M. A. Kilmon, N. J. Wagner, A. L. Garland, L. Lin, K. Aviszus, L. J. Wysocki, and B. J. Vilen Macrophages prevent the differentiation of autoreactive B cells by secreting CD40 ligand and interleukin-6 Blood, September 1, 2007; 110(5): 1595 - 1602. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Gilbert, D. G. Carnathan, P. C. Cogswell, L. Lin, A. S. Baldwin Jr, and B. J. Vilen Dendritic Cells from Lupus-Prone Mice Are Defective in Repressing Immunoglobulin Secretion J. Immunol., April 15, 2007; 178(8): 4803 - 4810. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Fujita, M. Yoshizumi, Y. Izawa, N. Ali, H. Ohnishi, Y. Kanematsu, K. Ishizawa, K. Tsuchiya, and T. Tamaki Transactivation of Fetal Liver Kinase-1/Kinase-Insert Domain-Containing Receptor by Lysophosphatidylcholine Induces Vascular Endothelial Cell Proliferation Endocrinology, March 1, 2006; 147(3): 1377 - 1385. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. A. Patel, A. Longacre, K. Hsiao, H. Fan, F. Meng, J. E. Mitchell, J. Rauch, D. S. Ucker, and J. S. Levine Apoptotic Cells, at All Stages of the Death Process, Trigger Characteristic Signaling Events That Are Divergent from and Dominant over Those Triggered by Necrotic Cells: IMPLICATIONS FOR THE DELAYED CLEARANCE MODEL OF AUTOIMMUNITY J. Biol. Chem., February 24, 2006; 281(8): 4663 - 4670. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Fan, V. A. Patel, A. Longacre, and J. S. Levine Abnormal regulation of the cytoskeletal regulator Rho typifies macrophages of the major murine models of spontaneous autoimmunity J. Leukoc. Biol., January 1, 2006; 79(1): 155 - 165. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Longacre, J. S. Koh, K. K-H. Hsiao, H. Gilligan, H. Fan, V. A. Patel, and J. S. Levine Macrophages from lupus-prone MRL mice are characterized by abnormalities in Rho activity, cytoskeletal organization, and adhesiveness to extracellular matrix proteins J. Leukoc. Biol., November 1, 2004; 76(5): 971 - 984. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Fan, A. Longacre, F. Meng, V. Patel, K. Hsiao, J. S. Koh, and J. S. Levine Cytokine Dysregulation Induced by Apoptotic Cells Is a Shared Characteristic of Macrophages from Nonobese Diabetic and Systemic Lupus Erythematosus-Prone Mice J. Immunol., April 15, 2004; 172(8): 4834 - 4843. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Drobnik, G. Liebisch, F.-X. Audebert, D. Frohlich, T. Gluck, P. Vogel, G. Rothe, and G. Schmitz Plasma ceramide and lysophosphatidylcholine inversely correlate with mortality in sepsis patients J. Lipid Res., April 1, 2003; 44(4): 754 - 761. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. K. Potter, J. Cortes-Hernandez, P. Quartier, M. Botto, and M. J. Walport Lupus-Prone Mice Have an Abnormal Response to Thioglycolate and an Impaired Clearance of Apoptotic Cells J. Immunol., March 15, 2003; 170(6): 3223 - 3232. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Bouzahzah, S. Jung, and J. Craft CD4+ T Cells from Lupus-Prone Mice Avoid Antigen-Specific Tolerance Induction In Vivo J. Immunol., January 15, 2003; 170(2): 741 - 748. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Reddy, K.-H. K. Hsiao, V. E. Abernethy, H. Fan, A. Longacre, W. Lieberthal, J. Rauch, J. S. Koh, and J. S. Levine Phagocytosis of Apoptotic Cells by Macrophages Induces Novel Signaling Events Leading to Cytokine-Independent Survival and Inhibition of Proliferation: Activation of Akt and Inhibition of Extracellular Signal-Regulated Kinases 1 and 2 J. Immunol., July 15, 2002; 169(2): 702 - 713. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. H. S. Kabarowski, K. Zhu, L. Q. Le, O. N. Witte, and Y. Xu Lysophosphatidylcholine as a Ligand for the Immunoregulatory Receptor G2A Science, July 27, 2001; 293(5530): 702 - 705. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
