| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Rheumatology Section, Evans Memorial Department of Clinical Research and Department of Medicine, Boston University Medical Center, Boston, MA 02118
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
) from young,
prediseased, lupus-prone MRL/++ and New Zealand Black/White
F1 mice display defective production of TNF-
, IL-1, and
IL-6, but normal production of IL-10. In an attempt to determine the
potential functional implications of this phenotype for autoimmunity,
we demonstrate here that endotoxin-activated M from these
lupus-prone mice showed dramatically reduced expression of IL-12, a
cytokine essential for Th1 responses that may be defective during
lupus. IL-12 production was also reduced by M from the control
BALB/c strain, compatible with the concept that a genetically
programmed deficit in IL-12 levels may underlie the IL-4-dominated
BALB/c response to infection by the parasite Leishmania
major. Although both IL-12 and TNF- expression defects by
M from lupus-prone strains are expressed rapidly after activation,
treatment with each cytokine demonstrated that only TNF- contributes
to the subsequent dysregulation of M IL-1 and IL-6 expression in
these strains, and that the reduced autocrine activity of defective
IL-12 or TNF- levels was not causal to each other. Although the
intrinsic defect in IL-12 expression by lupus-prone and BALB/c M may
lead to defective Th1 responses, these M responded to the
Th1-derived cytokine, IFN-
, in a normal fashion suggesting a
defective role in the induction, rather than the propagation, of Th1
responses in these mice. Our finding of a conserved intrinsic defect in
IL-12 production by M from the two principal mouse models of
multigenic lupus provides insight into how excessive humoral responses
may develop, and perhaps be prevented, in systemic autoimmune
disease. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Fundamental to understanding the initiation of autoimmune disease is the determination of why the control of autoreactive lymphocyte function is not established. One critical issue to address is the extent to which lymphocyte defects are truly intrinsic, or, alternatively, result secondarily from defects in the function of APCs or other regulatory cell populations. Evidence exists for both of these positions. Evidence supporting intrinsic B cell defects has come primarily from studies using MRL/lpr mice (3, 4). Because these mice display aberrant, nonfunctional Fas proteins, all cell populations in MRL/lpr mice whose growth and function are regulated by Fas-induced apoptosis are likely to be affected. The extent to which mice bearing the lpr mutation provide insight into the basis of multigenic human autoimmunity remains to be determined. The demonstration of B cell hyperactivity in young New Zealand Black/White (NZB/W) F1 and BxSB lupus-prone mice, or the transfer of T cell-depleted bone marrow from these mice into SCID mice (5, 6), has suggested that B cells from these multigenic lupus models may also have intrinsic defects in function. While such experiments emphasize the early development of aberrant B cell function in lupus, they do not substantiate that defective function is intrinsically programmed within the B cell, and the importance of accessory cells in permitting expression of defective B cell function has been demonstrated (7).
Studies focusing on the function of regulatory cells, such as
macrophages (M) (8, 9, 10, 11, 12, 13, 14, 15), from the best-studied murine lupus models,
MRL/++ and NZB/W F1, have revealed dramatic defects in
production of the proinflammatory cytokines TNF- (12, 15), IL-1
(8, 9, 10, 15), and IL-6 (15), but not the anti-inflammatory cytokine
IL-10 (15). Of these, aberrant IL-1 expression has been shown to be
truly intrinsic, i.e., independent of the contribution of other cell
types (9, 10). It is noteworthy that the onset of defective production
of the individual proinflammatory cytokines occurs sequentially, and
moreover, that the reduced autocrine stimulation caused by the TNF-
defect appears to be responsible for the subsequent IL-1 and IL-6
defects (15). Nevertheless, each of these events is triggered in and
controlled by M alone and is thus intrinsic to the M. These
studies have led us to question the potential functional contribution
of M to B cell hyperactivity in these mice. This contribution could
be both direct, on the B cell itself, and indirect, via modulation of T
cell function. To address these issues, we first extended our
characterization of M from MRL/++ and NZB/W F1 mice to
include the immunomodulatory cytokine, IL-12, which is mainly produced
by M and is critical for directing development of the Th1 responses
(16) that may be defective in lupus-prone mice (17). Our results show
that production of IL-12 is dramatically reduced in M from both
MRL/++ and NZB/W F1 mice, which appears to be similar in
nature to that of TNF- but not the subsequent IL-1 and IL-6 defects.
In addition, M IL-12 production in response to the Th1-derived
cytokine, IFN-, was normal in these lupus-prone strains, suggesting
a defect in the initiation, but not the propagation, of Th1 responses
in the disease process. This study provides additional insights into
the nature of the cytokine defects that uniquely characterize the two
principal models of multigenic lupus and suggests that the
cytokine-dependent regulation of the Th1 subset by the cells of the
innate immune system may be an important event in the establishment of
lupus.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Four-week-old BALB/c, A/J, C57BL/6, C57BL/10, DBA/2, C3H/OuJ, MRL/++, and NZB/W F1 male mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were maintained for 1 wk after arrival in a pressurized (one-way flow) room.
Reagents
Recombinant murine (rMu)TNF- (107 U/mg; cat. no.
19321T) was purchased from PharMingen (San Diego, CA), rMuIFN-
(107 U/mg) was kindly provided by Genentech (South San
Francisco, CA), and rMuIL-12 (3 x 106 U/mg) was a
gift from Dr. Victor H. Van Cleave (Genetics Institute, Cambridge, MA).
LPS (Escherichia coli: 0111:B4; Sigma, St. Louis, MO) was
stored at -70°C (2 mg/ml in H2O) and diluted immediately
before use. RPMI 1640 medium with glutamine (cat. no. 12-702F) was
supplemented with 0.5% HEPES (cat. no. 17-737), 1%
penicillin/streptomycin solution (cat. no. 17-602E), and 5% FBS (all
components of medium from BioWhittaker, Walkersville, MD) and was used
for culturing M. HBSS (BioWhittaker) was used for cell washing.
M isolation and culturing
Peritoneal exudate M were obtained by peritoneal lavage with
cold HBSS 4 days after a 2 ml i.p. injection of 4% thioglycollate
broth (Remel, Windsor, CT; cat. no. 07178). Cells were pooled from at
least three mice per strain, washed, resuspended in fresh medium, and
seeded at 105 cells in 100 µl per well of 96-well flat
bottom tissue culture-treated plates (Costar, Cambridge, MA; cat. no.
3596). Cells were incubated for 2 h at 37°C, 5%
CO2, in a humidified chamber to allow M to adhere and
spread. Nonadherent cells were removed by adding 200 µl of warmed
HBSS to each well, resuspending nonadherent cells by moderately tapping
the plate, and flicking the plate to discard the nonadherent cells.
This washing was performed three times, after which 50 µl of medium
was immediately added to each well. Remaining cells were >98% M,
as assessed by morphologic examination and nonspecific esterase
staining. Although 
85% of the total exudate cell population were
adherent M within each strain, to ensure that equal numbers of
adherent M among strains remained after washing, nonadherent cells
from washes of single wells from each strain were routinely counted and
showed no significant differences among strains. Cytokines, Abs, LPS,
or medium were added to each well to yield a final volume of 200 µl.
Conditioned medium was collected in a sequential fashion (e.g., 0–16,
16–24, 24–36 h), with change of medium and fresh LPS along with other
factors at each time point, and stored at -20°C for assessment of
cytokines. This method of conditioned medium collection has the
advantage of permitting measurement of cytokine levels independent of
levels produced during an earlier period, a feature that contributes to
accurate assessment of changes in the kinetic pattern of cytokine
production. The three culturing periods used for the kinetic assessment
of cytokine correlate to an early, intermediate, and late period that
collectively span the entire time during which most of the cytokine is
produced. For example, >90% of the total amount of IL-12 is produced
within 24 h, and the rest is produced within 24–36 h (see Fig. 2
;
see Ref. 15 for kinetics of other cytokines).
|
IL-12 levels in M-conditioned medium were assessed by a
MuIL-12-specific ELISA, a gift from Dr. Victor H. Van Cleave. The
general cytokine ELISA protocol developed by PharMingen was used.
Briefly, two rabbit polyclonal anti-MuIL-12 Abs preparations,
R03B03 (3 µg/ml) and B03B02 (biotinylated, 1:500 dilution), that were
prepared by immunizing rabbits with the IL-12 heterodimer (p70) were
used for capture and detection, respectively, along with avidin-labeled
peroxidase (Sigma, cat. no. A-3151) and 2,2'-azino-bis (3-ethyl
benzthiazoline-6-sulfonic acid) solution (Sigma, cat. no.
A-1888) for development. One hundred microliters of undiluted
M-derived conditioned medium was added to the ELISA, and rMuIL-12
was used to generate a standard curve. IL-6 and IL-10 levels in M
supernatants were assessed by ELISA (mAb from PharMingen; anti-IL-6
mAb, cat. nos. 18071D and 18082D, anti-IL-10 mAb, cat. nos. 18141D
and 18152D). IL-1 bioactivity in M lysates was measured in the
D10.G4 bioassay as previously described (15, 18), and TNF- levels in
M conditioned medium were assessed using the WEHI-164 bioassay as
previously described (15, 19). One unit of cytokine activity in
bioassay is defined as the amount of cytokine that caused half maximum
cell viability as assessed with Alamar Blue solution (AccuMed
International, Westlake, OH; cat. no. 00–100).
Statistical analysis of data
All means and SE in figures were calculated from triplicate values and the Student’s t test was used to compare mean values.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
from MRL/++ and NZB/W
F1 mice
To evaluate one possible mechanism for aberrant M cytokine
production contributing to the dysregulation of lymphocyte function in
lupus, we investigated whether M from lupus-prone strains aberrantly
produced IL-12. This cytokine is known to play a dominant role in the
appropriate development of Th1 cells, in establishing the balance of
Th1 and Th2 populations in normal individuals, and in triggering the
divergence from this balance that appears to be a critical component of
the development of autoimmune disease (16, 17). Peritoneal M were
obtained from young (4-wk-old), prediseased, male MRL/++ and NZB/W
F1 mice and from five age-matched control murine strains,
BALB/c, A/J, C57BL/6, DBA/2, and C3H/OUJ. M were activated
with LPS for 16 h, and culture-conditioned medium was assessed for
IL-12 levels by ELISA (Fig. 1). The data
reveal that M from MRL/++ and NZB/W F1 strains are
dramatically impaired and produce 10- to 20-fold less IL-12 than all
control strains, except BALB/c. Activation of M with other doses of
LPS (i.e., 10 ng/ml and 1000 ng/ml) resulted in defects of similar
magnitude in M from the lupus-prone strains (data not shown).
Interestingly, BALB/c M produce notably less IL-12 (4-fold less)
than M from the other control strains. Thus, the BALB/c strain,
which is known to have defective development of a protective Th1 type
response during some parasitic infections (i.e., Leishmania
major; reviewed in 20 , is here shown to have substantially
impaired production of the Th1-inducing cytokine, IL-12. The
association in BALB/c mice of low IL-12 levels with restrictions in
selected Th2-dependent immune responsiveness suggests that the similar,
if not more pronounced, IL-12 defect in the MRL/++ and NZB/W
F1 strains (in nine experiments, MRL/++ and NZB/W
F1 M produced IL-12 levels at an average of 62% ± 10%
(p < 0.01) and 70% ± 12%
(p < 0.01), respectively, of those of the
BALB/c M during the first period) may well have physiological
relevance to the immune dysregulation in these strains.
|
from C57BL/6 mice were selected
as a representative control strain because they routinely produced
levels of IL-12 close to the median value for normal strains. M from
MRL/++ and NZB/W F1 mice consistently displayed at least a
10-fold reduction in IL-12 production relative to C57BL/6, regardless
of the period of evaluation (Fig. 2
A). BALB/c M, as noted
earlier, produced substantially less IL-12 than M from any other
normal strain. Defective IL-12 expression by M from lupus-prone
strains is not a consequence of a reduction in either viability or
metabolic activity and does not reflect a broad dysregulation of M
function inasmuch as viability, protein synthesis, and induction of
LFA-1 and IL-10 all do not significantly vary between M from control
and lupus-prone strains (Refs. 15 and 21, and data not shown). These
results show that the defective M cytokine production profile in the
MRL/++ and NZB/W F1 strains, originally defined as
encompassing IL-1, IL-6, and TNF-, but not IL-10 (15), can
now be extended to include IL-12 as well.
Lupus-prone M defects in IL-12 and TNF- production do not
impact each other
Defective TNF- production by M from lupus-prone mice is
manifested soon after stimulation (0–12 h), and clearly before
defective expression of IL-6 (12–24 h) or IL-1 (48 h) (15). In fact,
compromised autocrine stimulation by TNF- in MRL/++ and NZB/W
F1 M appears to cause the subsequent defects in IL-1 and
IL-6 production (15). Therefore, we addressed whether the early TNF-
defect might contribute in a similar manner to the dramatically reduced
IL-12 expression in these M or, conversely, whether defective IL-12
might regulate TNF- expression. M from control and lupus-prone
mice were activated with LPS in the presence or absence of a
physiological concentration of TNF- (1000 U/ml, a level routinely
produced by control M under these conditions (15)). Conditioned
medium was collected at 16 h and assessed for IL-12 (Fig. 2, A and B). TNF- did not stimulate LPS-activated
IL-12 production by M from any strain during any interval, and,
thus, it is unlikely that the defective TNF- production
characteristic of M from lupus-prone strains contributes to their
IL-12 defect (Fig. 2B). Similarly, the addition of a
physiologically high concentration (10 ng/ml; see Figs. 1 and 2) of
IL-12 to LPS-activated M had no substantial effect on TNF-
expression, and, therefore, IL-12 is unlikely to be responsible for the
defective TNF- production by MRL/++ and NZB/W F1 M
(Fig. 3). In fact, IL-12 treatment tended
to modestly down-regulate TNF- production in M from all strains
tested, except A/J, where inhibition of M IL-12 production was
routinely 50–60% (Fig. 3).
|
, nor did it affect the dramatic
ability of TNF- to correct the progressive MRL/++ M defect in
IL-6 production that occurs after 24 h (Fig. 4). Similar results were obtained with
M from other control strains and from NZB/W F1 mice
(data not shown). IL-12 also had no effect on either normal M IL-1
expression, or on the defective kinetic production of IL-1 by M from
lupus-prone strains (data not shown). These findings further
demonstrate that IL-12 does not substantially modulate expression of
these proinflammatory cytokines, and more importantly, that defective
IL-12 is not involved in dysregulated expression of the other
proinflammatory cytokines by M from lupus-prone strains. The
unresponsiveness of M to IL-12 is further demonstrated by the
observation that TNF- production by M from C57BL/6 mice, a strain
that produces low levels of TNF- (15) and substantial levels of
IL-12 (see Figs. 1 and 2), is not enhanced by the Ab-mediated
neutralization of endogenous IL-12 production (453 ± 50 U/ml with
LPS alone and 457 ± 44 U/ml with LPS plus 3 µg/ml
anti-IL-12 mAb; IgG1 isotype control was similarly without effect
on TNF- expression). Thus, defective expression of IL-1 and IL-6
(15), but not IL-12, arises as a consequence of the early defect in
TNF- expression, and, moreover, defective IL-12 expression does not
contribute to defective TNF-, IL-1, and IL-6 expression.
|
IL-12 production is stimulated by the Th1-derived
cytokine, IFN-, in a normal fashion
The compromised ability of lupus-prone M to produce
sufficient levels of IL-12 induced with mitogen alone suggests that
these M would produce suboptimal levels of IL-12 during the
initiation of a Th1 response. IL-12 is required for the initial
production of the Th1-derived cytokine, IFN-, which in turn
amplifies the development of Th1-responsive cells by up-regulating M
IL-12 production; therefore, we determined whether lupus-prone
M were appropriately responsive to IFN- in their IL-12
production, and thus, would be capable of promoting an ongoing Th1
response. To determine the degree of responsiveness to IFN- by M
from lupus-prone strains, M from control and lupus-prone mice were
activated with LPS in the presence or absence of IFN-, and
conditioned medium was then assessed for IL-12 (Fig. 5A). The greatest augmentation
(roughly 20-fold) of IL-12 production by IFN- treatment occurred in
M from the two lupus-prone strains and the normal BALB/c strain.
Strikingly, IFN- up-regulated IL-12 production by LPS-activated M
from all control strains in a strictly-defined manner that was
inversely proportional to IL-12 levels induced by LPS alone (Fig. 5B). Additionally, this response is not simply due to a
shift in kinetics because M from lupus-prone mice continued to
demonstrate an enhanced responsiveness to IFN- during the
intermediate and late intervals, and in response to other doses (0.1 or
10 ng/ml) of IFN- as well (data not shown). Interestingly, the low
levels of IL-12 produced by the BALB/c strain were restored to normal
levels by IFN- treatment, but unlike those of lupus-prone strains,
were enhanced to precisely the extent predicted by the relative
differences among the other control strains (Fig. 5B). A
likely interpretation of these data is that IFN--signaling is intact
in both lupus-prone and BALB/c mice, but the BALB/c strain, although a
low IL-12 producer upon stimulation with LPS alone, defines the lower
limit of the normal cytokine production spectrum because IL-12 levels
were efficiently restored by IFN- to those of all other normal
strains. However, lupus-prone strains display a truly "defective"
pattern of IL-12 expression stimulated with LPS alone because their
response to IFN-, although intact and operating maximally, did not
restore IL-12 levels to those of normal strains. Nevertheless, the
dramatic response to IFN- by lupus-prone (and by BALB/c) M, which
led to IL-12 levels that approached those of normal strains, suggests
that these M would be capable of propagating, but not initiating, a
Th1 response.
|
distinguishes the regulation of IL-1, IL-6, and
IL-10 production from that of IL-12 and TNF-
The IL-12 and TNF- defects may arise from common mechanisms
inasmuch as they show similar kinetics (i.e., early expression, see
Figs. 1–3, and 15 , expression of one cytokine does not appear to
modulate expression of the other, and the extent of their defects is
dramatically reduced by IFN--treatment (see Ref. 15 for IFN-
regulation of defective TNF- expression). Conversely, the diminished
autocrine stimulation attributable to early defective TNF-
expression (15), and not, as shown here, to the early defective IL-12
production (see Figs. 3 and 4), appears responsible for the downstream
IL-1 and IL-6 expression defects in MRL/++ and NZB/W F1
M. Thus, while at least part of the mechanism leading to the
downstream IL-1 and IL-6 defects has been revealed, the underlying
aberrant events responsible for defective TNF- and IL-12 expression
remained to be determined. Moreover, the regulation of these cytokines
by IFN- also distinguishes two classes of cytokine defects (Table I). Production of TNF- and IL-12 is
strongly stimulated by IFN- (see Fig. 5 and Ref. 15, respectively),
whereas IL-1 and IL-6 production are either suppressed or not
substantially modulated, respectively, by IFN- treatment (Table I).
Other doses of IFN- (i.e., 0.1 and 10 ng/ml) demonstrated similar
modulation of IL-1 and IL-6 production in a dose-dependent fashion
(data not shown). These data demonstrate that the two classes of
cytokine defects, 1) the early onset defects of TNF- and IL-12, and
2) the later onset, or progressive, defects of IL-1 and IL-6, can be
further distinguished by their initial response to IFN- in that the
earliest aberrant functional events in M from lupus-prone strains
appear to compromise the expression of cytokines that can be strongly
stimulated by IFN-. We next tested, as a corollary to this finding,
whether cytokines that are not dysregulated in lupus-prone M might
also be refractory to IFN--mediated stimulation. IFN- treatment
did not stimulate and, in fact, suppressed IL-10 production by
LPS-stimulated M from control and lupus-prone strains (Table I),
supporting the contention that only cytokines stimulated during early
periods by IFN- (i.e., IL-12 and TNF-) show early defective
expression.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production of at least three proinflammatory
cytokines, TNF-, IL-1, and IL-6, that may be fundamental to the
disease process (9, 10, 15). In addition, M from both strains are
also characterized by normal IL-10 expression, and by the hierarchy of
TNF- that, by virtue of its under-expression, causes the subsequent
defects in IL-1 and IL-6 (15). That the defects are readily apparent in
M obtained from young, prediseased mice, are common to both
lupus-prone strains, and are intrinsic in nature, suggest that aberrant
expression of these immunomodulatory cytokines may play a critical role
in the initiation and development of the disease process. Indeed, the
critical contribution of these defects in M-derived cytokines has
been demonstrated by the therapeutic effect of administration of
TNF- or IL-1 to young, lupus-prone, NZB/W F1 mice before
the onset of disease (12, 27, 28). Here, we demonstrate a novel and
dramatic defect in MRL/++ and NZB/W F1 M production of
IL-12, a cytokine that is critical in the development of Th1 responses
(16). This IL-12 defect was manifested in a manner similar to that of
TNF- (15) in that both defects developed rapidly after activation
with LPS and could be substantially normalized by treatment with
exogenous IFN-. However, defective expression of IL-12, unlike that
of TNF- (15), did not contribute to the subsequent IL-1 and IL-6
defects in M from lupus-prone mice because addition of rMuIL-12 (or
the addition of anti-IL-12 mAb) did not substantially regulate IL-1
or IL-6 production. Furthermore, the IL-12 and TNF- defects did not
contribute to each other because addition of either cytokine had no
substantial regulation of expression of the other. These findings
strengthen the premise that cytokine dysregulation defines a conserved
and perhaps dominant pathway leading to systemic autoimmunity in those
strains whose disease, like human lupus, is multigenic in origin.
Earlier we had reported evidence for two distinct classes of cytokine
defects expressed in both MRL/++ and NZB/W F1 M (15).
These are defined operationally by their time of expression after M
stimulation: expression of the TNF- defect preceded expression of
the defects in IL-1 and IL-6, and, moreover, caused the latter defects.
The IL-12 defect reported here falls into the former category based on
kinetics, yet is distinct from TNF- in that it neither precipitated
nor modulated the ensuing defects in IL-1 and IL-6. The two classes of
cytokine defects defined above can be further distinguished by their
responsiveness to IFN-, an NK and T cell-derived cytokine known to
enhance M cytokine production (29). IFN- treatment dramatically
stimulated LPS-induced IL-12 and TNF- production (15) by M from
all strains, and the response was noted to be inversely proportional to
the basal level of cytokine produced upon stimulation with LPS alone.
Conversely, IFN- suppressed the early (16 h) production of IL-1 and
did not substantially regulate IL-6 expression, these targets being
representative of the second class of cytokine defects. This selective
inhibitory activity of IFN- on IL-1 (as well as on IL-10), and its
lack of regulation of IL-6 production, have also been reported by
others (30, 31). It is reasonable that the cytokine defects of the
first class (i.e., TNF- and IL-12) are caused by one or more
underlying aberrant events that can be overcome or normalized by
IFN--induced signals that normally stimulate those cytokines. These
stimulatory signals arising from the IFN- receptor may include both
secondary messenger molecules, such as JAK 1 and 2, and STAT1
molecules, and several transcription factors (32) and may provide clues
as to the aberrant molecular events that arise in these strains.
Although IFN- dramatically suppressed LPS-induced production of the
autocrine inhibitory cytokine IL-10, it is unlikely that IFN-
reduced the extent of defective TNF- and IL-12 expression in MRL/++
and NZB/W F1 M solely by suppressing IL-10 for the
following reasons: 1) The autocrine inhibitory activity of IL-10 does
not appear to be involved in defective TNF- (15) or IL-12 production
(our unpublished observation) because Ab-mediated neutralization
of IL-10 did not reduce the extent of these defects. 2) LPS-activated
M from MRL/++ and NZB/W F1 strains do not over-express
IL-10 (15). 3) The IFN--induced augmentation of TNF- (15) and
IL-12 production among control M is proportional to their IL-10
levels (15), suggesting that IFN- may normalize TNF- and IL-12
production by inhibiting IL-10; however, clearly the TNF- and IL-12
response to IFN- by M from lupus-prone mice is not proportional
to their IL-10 levels (i.e., MRL/++ have lower IL-10 and NZB/W
F1 have higher IL-10 levels than BALB/c (15)). Therefore,
IFN- appears to normalize TNF- and IL-12 expression in MRL/++ and
NZB/W F1 M by an IL-10-independent mechanism.
These findings suggest the possibility that the aberrant expression of
TNF- and IL-12 could develop as a consequence of an up-stream defect
in IFN- expression by these LPS-activated M. There is evidence
that LPS-activated murine peritoneal M express minute levels of
IFN- mRNA along with intracellular, but not extracellular,
expression of translated IFN- (33). Although it was suggested that
intracellular IFN- may cooperate with LPS to induce TNF-
expression (33), and while we have confirmed M IFN- production by
RT-PCR and Southern blot analysis (our unpublished observation), we did
not find defective expression of LPS-activated IFN- mRNA levels in
M from lupus-prone strains by these procedures (our unpublished
observation), nor could we detect secreted IFN- by ELISA, or
modulate TNF- or IL-12 production with anti-IFN- mAb
treatment (our unpublished observation). These results suggest that the
mechanisms underlying dysregulated TNF- and IL-12 production do not
include dysregulated endogenous IFN- production.
Defective expression of IL-12, in cooperation with defective TNF-
and IL-1, by M from lupus-prone mice may have an impact on
disease both during and after initiation of the disease process. The
initial polyclonal hyperexpansion of ubiquitous autoreactive
lymphocytes that appears as the first stage of disease (1, 2) may be
linked to defective TNF--induced apoptosis in addition to diminished
suppressor activity by IFN-, an NK- and T cell-derived cytokine
normally stimulated by the combination of TNF-, IL-1, and IL-12
(34, 35, 36), all of which we have found to be defectively produced by
MRL/++ and NZB/W F1 M (17). Following this polyclonal
hyperexpansion of autoreactive lymphocytes, defective IL-12 expression
may also permit the development of the strong, Ag-specific Th2-mediated
humoral responses, such as those directed toward chromatin (24), that
characterize later stages of lupus. Indeed, administration of IL-12
blocks pathogenic autoantibody production and end-stage renal pathology
in lupus-like, mercury-induced autoimmune disease in mice (37).
Moreover, that IL-12 administration to cultures of PBMC from human SLE
patients directly suppresses the excessive spontaneous production of Ig
and anti-DNA IgG Ab (38), along with the reduced IL-12 and IFN-
levels produced by these cultures (39, 40), suggests that defective
IL-12 levels in human SLE leads to hypergammaglobulinemia and the
generation of pathogenic Ab. Furthermore, it has recently been
reported, using a T cell receptor transgenic mouse, that M-derived
TNF- and IL-1 are required for IL-12 to induce development of a Th1
population from naive precursors in vitro (41). Thus, the defective
TNF- and IL-1 expression by M from lupus-prone strains, in
concert with defective IL-12 production, could play a fundamental role
in lupus by fostering the polyclonal hyperexpansion of autoreactive
lymphocytes and the subsequent establishment of a Th1-deficient
environment, which, ultimately, would lead to enhanced production of
pathogenic autoantibodies.
The physiological relevance of these findings will be determined, in
part, by whether the extent of defective IL-12 expression noted in the
lupus-prone strains is sufficient to alter the balance of T cell
subsets or otherwise promote pathogenic Ab responses. In this light, it
is noteworthy that the low IL-12 expression by M from BALB/c mice
shown here correlates with the sensitivity of this strain to infection
by the parasite, L. major (20). BALB/c mice are unable to
lodge a protective Th1 response against L. major infection,
and this is associated with exuberant IL-4 production and a strong Th2
response (20). Conversely, C57BL/6 and C3H/OuJ strains, whose M
produce dramatically more IL-12 than do BALB/c M, are resistant to
infection and mount a strong Th1 response against this parasite. These
findings thus suggest a genetic basis for BALB/c susceptibility to
L. major infection linked to deficient IL-12 production.
Indeed, it has been demonstrated, using L. major-resistant
strains created from congenic back-crosses of BALB/c and C57BL/6
strains, that disease susceptibility of BALB/c mice segregated with a
region of chromosome 11 that contains the IL-12 (p40) gene (42). If the
low levels of IL-12 produced by BALB/c M do contribute to enhanced
IL-4 expression and L. major susceptibility in this strain,
then the more dramatically reduced IL-12 levels in the MRL/++ and NZB/W
F1 strains are likely to be of similar, or even greater,
physiological significance, and as such, may contribute to
autoimmunity. All the cytokine defects that we have reported are found
in both MRL/++ and NZB/W F1 M, and, moreover, are
expressed before any detectable disease signs. Thus, it is tempting to
speculate that the two defective pathways discussed earlier, 1) reduced
TNF-, leading to reduced IL-1 and IL-6, and 2) reduced IL-12, may
both be important for establishing lupus of multigenic origin.
Our findings demonstrate that defective M proinflammatory cytokine
production has been conserved among at least two genetically distinct
backgrounds prone to murine lupus, suggesting a common, intrinsic
cellular aberrancy that might be diagnostic for this disease and, more
importantly, may be involved in the initiation and propagation of the
disease process. Clearly, the contribution of the M defects
described here must be viewed in the context of the gene mapping
studies which have revealed multiple genetic linkage groups associated
with specific aspects of murine lupus (24). In particular, the elegant
genetic studies of Wakeland and coworkers (22, 24) and Kotzin and
coworkers (23) have revealed loci which are associated with B cell
hyperactivity. Although there are claims of intrinsic defects in B
lymphocytes function from lupus-prone strains, the splenic B cell
populations studied generally are the product of T cell depletion, and
thus enriched both for B cells and M (5, 6, 22). Neither percoll
enrichment of B cells nor adherence-based elimination of M are
particularly helpful procedures due to the marked ability of spleen
cells to generate M from immature, nonadherent precursors in culture
(our unpublished observations). While the use of purified long-term B
cell lines transferred into SCID mice provides more compelling support
for intrinsic B cell defects (6), these animals do not manifest all
aspects of NZB/W F1 B cell hyperactivity, in keeping with
the concept that the complexity of lupus is most likely due to several
mutations operating at different times in development or simultaneously
in different cells types. Our findings here place the M, among other
cells of the innate immune system, in a central position regarding the
initiation and development of autoimmunity. Moreover, they raise
questions as to whether defects ascribed to B and T cells in these
multigenic models of lupus (MRL/++, NZB/W F1) are intrinsic
to lymphocytes, or alternatively, reflect differences in lymphocyte
development or regulation that arise as a consequence of aberrant
control by accessory cells. It is anticipated that continued efforts to
understand the control and function of cells regulating innate immunity
will provide insight into the ways that dysregulated cytokine
expression can contribute, not only as a possible effector mechanism of
the end-stage inflammation of the disease process, but also to the
fundamental immune dysregulation that initiates the development of
lupus.
|
Acknowledgments |
|---|
(107 U/mg). Much gratitude is extended to Karen S. Alleva
for her assistance in preparing the manuscript and in laboratory
maintenance, and to Dr. John J. Alleva for his critical analysis of
data presentation. |
Footnotes |
|---|
2 Current address: Neurocrine Biosciences, Inc., 10555 Science Center Drive, San Diego, CA 92121-1102.
3 Address correspondence and reprint requests to Dr. David I. Beller, Boston University Medical Center, Department of Medicine, Rheumatology Section, E5, Boston, MA 02118-2393. E-mail address:
4 Abbreviations used in this paper: SLE, systemic lupus erythematosus; NZB/W, New Zealand Black/White; M, macrophages; rMu, recombinant murine.
Received for publication April 9, 1998. Accepted for publication August 14, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in murine autoimmune lupus nephritis. Nature 331:356.[Medline]
in autoimmune NZB/NZW F1 mice. Clin. Immunol. Immunopathol. 52:421.[Medline]
. Annu. Rev. Immunol. 15:749.[Medline]
suppresses IL-1 but not lipopolysaccharide-induced transcription of IL-1. J. Immunol. 144:2216.[Abstract]
receptor. Annu. Rev. Immunol. 15:563.[Medline]
in macrophages by lipopolysaccharide. Int. Immunol. 5:1383.
T cells for production of IFN- is mediated by bacteria via macrophage-derived cytokines IL-1 and IL-12. J. Immunol. 154:5832.[Abstract]
in immune cell regulation. J. Leukocyte Biol.
58:373.
and negatively with disease activity in SLE. Cytokine 10:148.[Medline]
and TNF- are required for IL-12-induced development of Th1 cells producing high levels of IFN- in BALB/c but not C57BL/6 mice. J. Immunol. 160:1708.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]
|
|
|
|
 |
|
 L. M. Maier, S. K. Howlett, K. M. Rainbow, J. Clark, J. M. M. Howson, J. A. Todd, and L. S. Wicker NKG2D-RAE-1 Receptor-Ligand Variation Does Not Account for the NK Cell Defect in Nonobese Diabetic Mice J. Immunol., November 15, 2008; 181(10): 7073 - 7080. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. E. Zielinski, S. N. Jacob, F. Bouzahzah, B. E. Ehrlich, and J. Craft Naive CD4+ T Cells from Lupus-Prone Fas-Intact MRL Mice Display TCR-Mediated Hyperproliferation Due to Intrinsic Threshold Defects in Activation J. Immunol., April 15, 2005; 174(8): 5100 - 5109. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Charoenvit, G. T. Brice, D. Bacon, V. Majam, J. Williams, E. Abot, H. Ganeshan, M. Sedegah, D. L. Doolan, D. J. Carucci, et al. A Small Peptide (CEL-1000) Derived from the {beta}-Chain of the Human Major Histocompatibility Complex Class II Molecule Induces Complete Protection against Malaria in an Antigen-Independent Manner Antimicrob. Agents Chemother., July 1, 2004; 48(7): 2455 - 2463. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Ma, K. Gee, W. Lim, K. Chambers, J. B. Angel, M. Kozlowski, and A. Kumar Dexamethasone Inhibits IL-12p40 Production in Lipopolysaccharide-Stimulated Human Monocytic Cells by Down-Regulating the Activity of c-Jun N-Terminal Kinase, the Activation Protein-1, and NF-{kappa}B Transcription Factors J. Immunol., January 1, 2004; 172(1): 318 - 330. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. B. Simpson, M. S. Mistry, R. A. Maki, W. Yang, D. A. Schwarz, E. B. Johnson, F. M. Lio, and D. G. Alleva Cutting Edge: Diabetes-Associated Quantitative Trait Locus, Idd4, Is Responsible for the IL-12p40 Overexpression Defect in Nonobese Diabetic (NOD) Mice J. Immunol., October 1, 2003; 171(7): 3333 - 3337. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Liu and D. I. Beller Distinct Pathways for NF-{kappa}B Regulation Are Associated with Aberrant Macrophage IL-12 Production in Lupus- and Diabetes-Prone Mouse Strains J. Immunol., May 1, 2003; 170(9): 4489 - 4496. [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]
|
|
|
|
|
|
J. Liu and D. Beller Aberrant Production of IL-12 by Macrophages from Several Autoimmune-Prone Mouse Strains Is Characterized by Intrinsic and Unique Patterns of NF-{kappa}B Expression and Binding to the IL-12 p40 Promoter J. Immunol., July 1, 2002; 169(1): 581 - 586. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. G. Alleva, E. B Johnson, F. M Lio, S. A Boehme, P. J Conlon, and P. D. Crowe Regulation of murine macrophage proinflammatory and anti-inflammatory cytokines by ligands for peroxisome proliferator-activated receptor-{gamma}: counter-regulatory activity by IFN-{gamma} J. Leukoc. Biol., April 1, 2002; 71(4): 677 - 685. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. R. B. Bastos, J. M. Alvarez, C. R. F. Marinho, L. V. Rizzo, and M. R. D'Imperio Lima Macrophages from IL-12p40-deficient mice have a bias toward the M2 activation profile J. Leukoc. Biol., February 1, 2002; 71(2): 271 - 278. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Koh, Z. Wang, and J. S. Levine Cytokine Dysregulation Induced by Apoptotic Cells Is a Shared Characteristic of Murine Lupus J. Immunol., October 15, 2000; 165(8): 4190 - 4201. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. D. Mills, K. Kincaid, J. M. Alt, M. J. Heilman, and A. M. Hill M-1/M-2 Macrophages and the Th1/Th2 Paradigm J. Immunol., June 15, 2000; 164(12): 6166 - 6173. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S M Aronica, A Dozier, P Fanti, and M Nazareth Altered bone marrow cell sensitivity in the lupus-prone NZB/W mouse: regulation of CFU-GM colony formation by estrogen, tamoxifen and thrombopoietin Lupus, May 1, 2000; 9(4): 271 - 277. [Abstract] [PDF]
|
|
|
|
 |
|
 M. L. Stoll and J. Gavalchin Systemic lupus erythematosus--messages from experimental models Rheumatology, January 1, 2000; 39(1): 18 - 27. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
