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1 Cardiovascular Institute of the University of Pittsburgh Medical Center Health System, 2 Department of Molecular Genetics and Biochemistry, and 3 Center for Biological Imaging, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Tumor necrosis factor (TNF)-
plays a key role in the pathogenesis of septic shock syndrome, and
myocardial TNF- expression may contribute to this pathophysiology.
We examined the myocardial expression of TNF--related cytokines and
chemokines in mice exposed to lipopolysaccharide (LPS) and tested the
effects of anti-TNF therapy on myocardial cytokine expression. Cytokine
mRNA levels were measured by RNase protection assay, and protein levels
in the plasma and myocardium were assessed by enzyme-linked
immunosorbent assays. LPS (4 µg/g body wt ip) induced marked cytokine
expression, including TNF-, interleukin (IL)-1
, IL-6, and
monocyte chemotactic protein (MCP)-1, in both the plasma and
myocardium. Pretreatment with adenovirus-mediated TNF receptor fusion
protein (AdTNFR1; 109 plaque-forming units iv) decreased
plasma cytokine levels. In contrast, whereas myocardial IL-1
expression was also suppressed, expression of IL-6 and MCP-1 was
not inhibited by AdTNFR1. In summary, anti-TNF treatment
differentially altered the cytokine expression in the plasma and
myocardium during endotoxemia. Inability to block myocardial expression
of IL-6 and MCP-1 suggests a possible mechanism for the failure of
anti-TNF therapies in the treatment of endotoxin shock.
adenovirus; chemokine; endotoxin shock; tumor necrosis factor-
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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SEPTIC SHOCK,
triggered by products of infectious agents, causes substantial
morbidity and mortality characterized by a hypotensive state
accompanied by inadequate tissue perfusion, metabolic acidosis, and
coagulopathy (6). Evidence suggests that tumor necrosis factor (TNF)-, which is a proinflammatory cytokine with pleiotropic biological effects (60), is a key mediator of the septic
shock syndrome induced by either lipopolysaccharide (LPS) or bacterial superantigens (6, 38, 40, 50). Indeed, in response to LPS,
TNF- is produced in large amounts earlier than any other cytokine
(6), and, given as a purified preparation, TNF- evokes most of the effects of LPS in animals including fever, shock, and death
(50). Furthermore, mice lacking TNF receptor type 1 (TNFR1) are resistant to septic shock (43, 45).
Given that TNF- plays a key role in the pathogenesis of septic shock
syndrome, anti-TNF therapy was expected to provide protection against
the toxicity of TNF- and reduce mortality. However, the results of
currently tested anti-TNF therapies for endotoxemia have been
controversial. In some (3, 17, 39) but not all (13,
33) experimental studies, anti-TNF therapies ablated TNF-
bioactivity and reduced mortality after LPS injection. Moreover, anti-TNF antibodies can convert a nonlethal model of endotoxemia into a
lethal one (12). In one clinical study (14),
administration of a monoclonal antibody to TNF- in patients with
septic shock limited hypotension. However, treatment with the TNFR:Fc
fusion protein in patients with septic shock failed to reduce
mortality, and higher doses appeared to be associated with increased
mortality (16).
Elevated plasma levels of TNF- have been reported in a variety of
cardiovascular diseases, including acute myocarditis (35), cardiac allograft rejection (8), myocardial infarction
(36), and congestive heart failure (32, 34),
as well as in endotoxemia (17, 51, 54). Recent studies
have demonstrated that the heart can produce TNF- in response to
pathological stresses (24, 49) and endotoxin (18,
23). In addition, transgenic mice overexpressing cardiac TNF-
develop myocardial hypertrophy and dilatation, interstitial infiltrates
and fibrosis, attenuation of adrenergic responsiveness, and robust
expression of a variety of TNF--responsive proinflammatory cytokines
and chemokines, including interleukin (IL)-1 and monocyte
chemotactic protein (MCP)-1 (29, 31). These observations
led us to hypothesize that the inability of anti-TNF therapy to improve
survival during endotoxemia is due to an inability to suppress the
expression of non-TNF- endotoxic-responsive cytokines and chemokines
in the heart.
To test this hypothesis, we neutralized TNF- bioactivity by
injecting mice with a replication-deficient recombinant adenovirus encoding a 55-kDa soluble TNF receptor (AdTNFR1) before LPS challenge. This anti-TNF treatment reduced the expression of TNF- and
TNF--responsive proinflammatory cytokines and chemokines in plasma
during endotoxemia but was ineffective in attenuating the myocardial
expression of the endotoxin-responsive cytokines IL-6 and MCP-1.
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MATERIALS AND METHODS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animal Preparation
Male 3-mo-old FVB mice (29.3 ± 1.8 g body wt) bred from an in-house colony were used for protocol 1. Male 3-mo-old TNFR1-deficient mice created on a C57BL/6 background (Jackson Laboratory) (43) were used for protocol 2. Age- and sex-matched wild-type mice served as controls. Detailed descriptions about the protocols are shown in Experimental Design. Animals were housed in cages at 20-22°C with a 12-h:12-h light-dark cycle. Animals were allowed free access to water and laboratory chow throughout the experimental period. The mice were utilized according to protocols approved by the Institutional Animal Care and Use Committee, University of Pittsburgh.Recombinant Adenoviruses
AdTNFR1 (26), encoding the extracellular domain of human 55-kDa TNFR coupled with a mouse IgG heavy chain (42), was used in the present study. The original viruses were generously provided by Dr. Bruce Beutler, University of Texas Southwestern Medical Center. The viruses were propagated in 293 cells, purified by cesium chloride density centrifugation, and stored in aliquots at
80°C as previously described (26). The
virus titer was equal to the optical density at 260 nm
(OD260) divided by 9.09 × 10
13
particles/ml. One hundred particles were assumed to be one
plaque-forming unit (pfu). After injection, the majority of virus is
extracted by the liver, with subsequent hepatic production and release
of soluble receptor into the peripheral circulation (26).
Intravenous injection of 109 pfu of AdTNFR1 has been shown
to inhibit TNF activity in plasma for up to 6 wk (26).
This dose of AdTNFR1 could effectively abrogate the changes of
myocardial inflammation and cardiac-specific gene expression in our
transgenic mice with cardiac-specific overexpression of TNF-
(29).
Experimental Design
The experimental design sought to examine whether treatment with soluble TNFR1 could ameliorate the expression of cardiac cytokines during endotoxemia in mice. To this end, we performed two experiments.Protocol 1.
Mice were divided into two groups as follows: 1) animals
injected with 109 pfu of AdTNFR1 through the retroorbital
venous plexi 1 wk before LPS exposure and 2) control animals
not injected with AdTNFR1. We used the LPS of Escherichia
coli 0127 (Sigma), which strongly stimulates TNF- production in
rat and human cardiomyocytes (55, 57). Animals were given
a single intraperitoneal injection of LPS (4 µg/g body wt) as a 1 µg/µl solution in physiological saline. At 0.5, 2, and 24 h
after LPS injection, the animals were euthanized. After the ventricular
weight was determined, excised ventricles were snap-frozen in liquid
nitrogen for RNA and protein analysis. In some of the animals
euthanized at 2 h after LPS challenge, hearts were perfused with
2% paraformaldehyde and then processed for immunohistochemical
analysis as described in Immunohistochemistry. Plasma was
also collected for assessment of cytokines and soluble TNFR1. The
hearts and plasma from age- and sex-matched mice exposed to saline
(instead of LPS) with or without AdTNFR1 pretreatment served as controls.
Protocol 2. TNFR1-deficient mice and wild-type controls were also injected with LPS the same way as protocol 1. Animals were divided into two groups, and each group was given either a low (4 µg/g body wt) or high (40 µg/g body wt) dose of LPS, respectively. At 2 h after injection, the animals were euthanized, and cardiac tissue and plasma samples were harvested as described in Protocol 1.
RNase Protection Assay
Total RNA was extracted from frozen tissues with the use of an acid guanidinium thiocyanate-phenol-chloroform method (9). The concentration of RNA in each sample was assessed spectrophotometrically. To evaluate transcript levels of cytokines in the myocardium, a commercially available multiprobe RNase protection assay kit (RiboQuant, PharMingen) was used, with the assay performed according to the manufacturer's protocol. Briefly, a set of 32P-labeled RNA probes synthesized from DNA templates using T7 polymerase was hybridized with 5 µg of total RNA, after which free probes and other single-stranded RNA were digested with RNases. The remaining RNase-protected probes were purified, resolved on denaturing polyacrylamide gels, and quantified by PhosphoImager using ImageQuant software (Molecular Dynamics). The value of each hybridized probe was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) included in each template set as an internal control (arbitrarily set as equal to 1). The following template sets for murine cytokines were used in the present study: mCK-2b (No. 45051P), IL-12p35, IL-12p40, IL-10, IL-1, IL-1, IL-1 receptor antagonist (Ra), IL-18, IL-6,
interferon (IFN)-
, macrophage migration inhibitory factor (MIF),
L32, and GAPDH; mCK-3b (No. 45071P), TNF-, lymphotoxin (LT)-,
TNF-, IL-6, IFN-, IFN-, transforming growth factor (TGF)-1, TGF-2, TGF-3,
MIF, L32, and GAPDH; and mCK-5 (No. 45026P), lymphotactin, regulated
upon activation normal T-cell expressed and secreted (RANTES), eotaxin,
macrophage inflammatory protein (MIP)-1, MIP-1, MIP-2,
IFN--inducible protein (IP)-10, MCP-1, T-cell activation gene
(TCA)-3, L32, and GAPDH.
Enzyme-Linked ImmunoSorbent Assay
Protein levels of cytokines were assessed using commercially available ELISA kits for mouse TNF-, mouse IL-1, mouse IL-6, mouse MCP-1, mouse IL-10, mouse IL-12p75, and human TNFR1 (Quantikine, R&D Systems). Plasma samples were measured at a dilution of
101-103 for TNF-, IL-1, IL-6,
and MCP-1 and 105-107 for TNFR1.
Cytokines in the myocardium were measured as previously reported
(30, 31). Briefly, frozen tissues (5-20 mg) were homogenized in 300-500 µl of ice-cold phosphate-buffered saline containing 1 mmol/l phenylmethylsulfonyl fluoride protease inhibitor (Sigma). After a brief centrifugation, samples were kept on ice for the
duration of the assay. Total protein levels were quantitated using a
commercially available assay (Bio-Rad Protein Assay, Bio-Rad Laboratories) with BSA (Sigma) as a standard. The same amount of
protein was applied for each immunoassay: 100 µg for TNF-, IL-1, IL-6, and MCP-1 and 1 µg for TNFR1. Cytokines provided by
the manufacturer were used as a standard. All assays were done in
duplicate. Results were analyzed spectrophotometrically at a wavelength
of 450 nm with a microtiter plate reader. The values are reported as
picograms or nanograms per milligram of protein for tissue samples and
nanograms or micrograms per milliliter for plasma samples.
Immunohistochemistry
To clarify which cell types of cardiac tissue are responsible for LPS-induced IL-6 production, we performed IL-6 immunostaining in some LPS-treated mice. Mouse hearts were perfused with 2% paraformaldehyde, followed by a 2-h postfix in the same fixative. After overnight cryoprotection in ice-cold 30% sucrose, the hearts were flash-frozen with liquid nitrogen-cooled isopentane. Cryostat (Microm) sections (6 µm) were cut and mounted onto Superfrost Plus slides (Fisher). Sections were then rinsed with PBS, followed by rinsing in a blocking buffer (0.5% BSA and 0.15% glycine in PBS) before treating with 5% normal goat serum for 30 min. Double-immunofluorescent detection was performed using a polyclonal rabbit anti-mouse IL-6 antibody (1:100, sc-7920, Santa Cruz) and a monoclonal rat anti-mouse CD45 antibody (1:100, 01111D, Pharmingen). Samples were treated with primary antibodies for 1 h at room temperature and then rinsed with blocking buffer. Fluorescent secondary antibodies included goat anti-rabbit IgG conjugated with CY3 (1:3000, Jackson ImmunoResearch Laboratories) and goat anti-rat IgG conjugated with CY5 (1:1000, Jackson ImmunoResearch Laboratories). Slides were treated with secondary antibodies for 1 h at room temperature and then sequentially rinsed with blocking buffer, followed by PBS before nuclei were labeled with Hoescht 33342 (Sigma). Slides were coverslipped and viewed with an Olympus Provis AX70 fluorescent microscope. Images were collected with a cooled charge-coupled device camera (Optronics Magnifier) at a 12-bit gray depth and assembled in Photoshop (Adobe). No further postprocessing or filtering of the images was performed.Statistics
The results are presented as means ± SD. Statistical comparisons were performed with the use of analysis of variance with Student-Newman-Keuls post hoc test. Differences were considered significant at a value of P < 0.05.| |
RESULTS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Body weight and ventricular weight of the animals at the time of death were comparable, with no significant differences among any of the groups (data not shown).
Adenovirus-Mediated Production of TNF Receptor Fusion Protein
To confirm that the dose of AdTNFR1 used in the present study (109 pfu iv) was adequate to block local bioactivity of TNF- in the myocardium, the plasma and myocardial levels of TNFR1 in
AdTNFR1-treated animals were assayed. As shown in Table
1, intravenous injections of
109 pfu of AdTNFR1 produced a substantial amount of TNFR1
in both the plasma and myocardial tissue after 1 wk of treatment.
Compared with the peak amounts of TNF- protein expressed in the
myocardium of LPS-treated mice, there was a large excess (~200-fold)
of TNFR1 protein in the heart. This amount of TNFR1 production is
comparable to that found in our previous study (29) with
TNF- transgenic mice in which AdTNFR1 treatment was effective in
reversing various pathophysiological changes induced by overexpression
of TNF-. Serum TNFR1 levels were similar at all time points after
LPS treatment.
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Expression of Cytokines in Myocardium in Response to LPS Administration
Because TNF- is known to induce the expression of other
proinflammatory cytokines and chemokines that contribute to
TNF--induced pathophysiology (60), we examined the
expression of a panel of cytokines using a multiprobe RNase protection
assay. Representative images of RNase protection assays are shown in
Fig. 1 and quantitative results are
summarized in Fig. 2. Intraperitoneal
injection of LPS induced the expression of a group of cytokines
(including TNF-, TNF-, IL-1, IL-1, IL-1Ra, IL-6, IL-10,
IL-12, IL-18, TGF-1, TGF-2,
TGF-3, and LT-) and a group of chemokines (including MCP-1, RANTES, MIP-1, MIP-1, MIP-2, eotaxin, and lymphotactin). In particular, the induction of TNF-, IL-1, IL-6, and MCP-1 expression in the myocardium in response to LPS challenge was robust.
In contrast, MIF was constitutively expressed in the myocardium in mice
without LPS treatment and was increased after LPS injection. Cytokines
IFN-, IFN-, IP-10, and TCA-3 were not detected in any samples
regardless of LPS treatment. The myocardial mRNA expression of various
cytokines were followed before LPS injection and at 0.5, 2.0, and
24 h after the injection. The maximal myocardial expression of
TNF- mRNA was observed 0.5 h after LPS treatment, whereas most
other cytokines showed peak responses 2.0 h after LPS treatment. A
significant exception was RANTES, which showed maximal expression
24 h after LPS injection (Fig. 1 and Table 1). Pretreatment with
AdTNFR1 partly but significantly reduced the myocardial mRNA expression
of IL-1 but did not change the expression of other cytokines,
including IL-6 and MCP-1. In addition, LPS treatment also resulted in
subtle but significant increase in cardiac IL-10 and IL-12p40 gene
expression, and these changes were not significantly affected by
AdTNFR1 (Fig. 2).
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To confirm that the changes in mRNA reflected alterations at the
protein level within the myocardium, we measured the protein levels of
six cytokines (TNF-, IL-1, IL-6, MCP-1, IL-10, and IL-12p75).
These cytokines were chosen because of their synergistic effects with
TNF- as well as their independent effects on cardiomyocyte function
and gene expression (7, 19, 25, 37, 44) or their
well-established roles in the pathogenesis of endotoxin shock
(21, 59). As shown in Fig.
3, myocardial TNF-, IL-1, IL-6, and
MCP-1 proteins were not found in mice without LPS treatment but were
abundant in the LPS-treated mice. One week of anti-TNF pretreatment
with soluble receptor led to a moderate but significant decrease in the
expression of IL-1 but not of IL-6 and MCP-1 proteins, consistent
with studies assessing levels of mRNA. In fact, anti-TNF treatment
actually increased the level of immunodetectable TNF- protein in the
myocardium. IL-10 and IL-12p75 protein were also significantly
increased by LPS treatment, although the amount of these two cytokines
was much less than the amount of the other four cytokines examined.
Cardiac IL-10 protein expression was significantly augmented by AdTNFR1
treatment, whereas IL-12p75 expression was not changed.
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LPS-induced cardiac cytokine expression was also examined in
TNFR1-deficient mice (TNFR1/). As shown in Fig.
4, cardiac expression of TNF-
in response to LPS was similar to or somewhat higher in TNFR1-deficient
mice compared with wild-type control mice (TNFR1+/+). In contrast, both
IL-1 and IL-6 expression were significantly less in TNFR1-deficient
mice. However, even in TNFR1-deficient mice, there were still
substantial increases in IL-1 or IL-6 expression, ~50% of that
reached in the wild-type LPS-treated mice.
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Circulating Cytokine Levels after LPS Administration
We also examined the plasma protein levels of these four cytokines in response to LPS injection (Fig. 5). TNF-, IL-1, IL-6, and MCP-1 proteins were not found in the plasma
of mice without LPS treatment but were abundant in the LPS-treated
mice, which is similar to that observed for myocardial expression. The
immunodetectable protein levels of plasma TNF- were
markedly increased by pretreatment with AdTNFR1. In contrast
to the result from myocardium, IL-1, IL-6, and MCP-1 proteins
induced by LPS were significantly decreased by AdTNFR1. AdTNFR1
pretreatment again significantly augmented LPS-induced IL-10
expression, whereas IL-12p75 levels were markedly decreased.
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Plasma cytokine levels in TNFR1-deficient mice after LPS
challenge are summarized in Fig. 6. The
increase of TNF- levels in TNFR1-deficient mice after LPS treatment
was significantly higher in TNFR1-deficient mice in both the low and
high dosages of LPS. However, plasma levels of both IL-1 and IL-6
concentration were markedly suppressed in TNFR1-deficient mice. These
observations were consistent with the previous report (45)
and our present studies using AdTNFR1.
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IL-6 Immunohistochemical Staining
Immunohistochemical staining of LPS-treated cardiac tissue was performed to determine the cell types that express IL-6 in response to systemic LPS challenge. Counterstaining with an anti-CD45 antibody was performed to distinguish cardiac infiltrating leukocytes from resident cardiac cells such as myocytes and fibroblasts. Representative images from LPS and saline-treated animals are shown in Fig. 7. Because both CD45-positive leukocytes and CD45-negative cardiac myocytes were positively stained with anti-IL-6 antibody in LPS-treated tissues, both of these cell types are likely sources of IL-6 production in response to LPS challenge.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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While TNF- has important direct biological functions and plays
a key role in the pathogenesis of endotoxin shock syndrome, it can also
induce the expression of "downstream" cytokines and chemokines that
may contribute to TNF--induced pathophysiology (60).
Furthermore, in endotoxin shock, different organs display diverse
patterns of cytokine expression (18, 52). In
addition, while endotoxin may induce the expression of substances
that alter cardiac function such as TNF- and other cytokines and
chemokines within the myocardium, endotoxin itself may have direct
cardiodepressant effects (41). The diversity of both
beneficial (3, 14, 17, 39) and antagonistic (12,
16) effects that may occur in endotoxemia consequent to
anti-TNF- therapies suggest a complex and incompletely understood
role of TNF- in this condition. One possible mechanism for the
divergent responses elicited by anti-TNF- therapy is that therapy
with monoclonal anti-TNF antibody attenuates activation of the
fibrinolytic system without influencing endotoxin-induced activation of
the coagulation system, generating a potential enhancing effect on
microvascular thrombosis in sepsis (53). In this study, we
suggest that relative to systemic effects measured by LPS-induced serum
cytokine levels, anti-TNF- therapy fails to completely protect the
myocardium from the detrimental effects elicited directly by LPS or
other cytokines induced by LPS through non-TNF-dependent mechanisms.
In this study, we utilized an anti-TNF therapy in which animals were
injected intravenously with an adenoviral vector expressing a fusion
protein of the extracellular domain of human TNFR1(p55) coupled with a
mouse IgG heavy chain (26). The virus is rapidly taken up
by the liver, which effectively becomes a production site for soluble
TNFR1. A single intravenous injection allows sustained expression of a
substantial amount of TNFR1 in the plasma, which permeates the
extracellular space of multiple organs (27) including the
heart, binds to TNF-, and limits its ability to interact with
cellular TNF receptors. The principle limitation of using recombinant
adenovirus is that endogenous immunological activity limits the ability
to reinject animals with AdTNFR1. Thus, after several months,
biologically significant levels of TNFR1 cannot be found in either the
plasma or tissues. However, as seen in our previous study, beneficial
biological effects of TNFR1 in mice overexpressing proinflammatory
cytokines can be clearly demonstrated within 2 wk of therapy and
persist through 6 wk (29). These results support the
efficacy of this approach in blocking the biological effects of TNF-
systemically. Despite the evidence that treatment with AdTNFR1 can
effectively block TNF- bioactivities in vivo, we cannot exclude the
possibility that a very low level of biologically active TNF- may
persist despite therapy with TNFR1, allowing low-level production of
downstream cytokines.
To better elucidate the differential expression of cytokines and
chemokines in the myocardium in response to LPS challenge with or
without anti-TNF- therapy, we used multiprobe RNase protection assay
panels to assess the gene expression of cytokines and chemokines in the
ventricle and ELISA to assess the protein expression of cytokines and
chemokines in the ventricle and plasma of the LPS-treated mice. The
principal finding of these studies is that, whereas LPS induces a
similar profile of cytokines and chemokines (TNF-, IL-1, IL-6,
IL-10, IL-12, and MCP-1) in the plasma and myocardium, plasma and
myocardial expression of these cytokines are differentially responsive
to anti-TNF therapy.
In the present study, intraperitoneal administration of LPS to mice
resulted in robust expression of a group of cytokines including
TNF-, IL-1, IL-6, and MCP-1 in both the myocardium and plasma.
Importantly, in the myocardium, there was a close correlation between
the measure of cytokine mRNAs and proteins. Pretreatment with soluble
TNF receptor partially but significantly reduced plasma levels of
IL-1, IL-6, and MCP-1. In contrast, anti-TNF pretreatment
significantly suppressed LPS-induced myocardial expression of IL-1
but had no effect on the production of IL-6 and MCP-1, suggesting that
the myocardial induction of these two cytokines occurs either directly
through LPS or through non-TNF--dependent pathways. To better assess
the role of TNF- in the cardiac response to LPS, we also treated
mice lacking functional TNFR1 protein (TNFR1/) with LPS. LPS still
induced an increase in plasma IL-1 and IL-6 levels, although at a
level significantly lower than that observed in their wild-type
littermates (TNFR1+/+), which is consistent with previous reports
(43, 45). In particular, plasma IL-6 levels were reduced
by ~80%, relative to those observed in LPS-injected TNFR1+/+ mice.
However, the cardiac expression of IL-6 in LPS-injected TNFR1/ mice
was still ~50% of that observed in TNFR1+/+ mice, suggesting that
the cardiac induction of IL-6 by LPS is not completely dependent on
signals mediated through the TNFR1 receptor. While we formally cannot
rule out the possibility that cardiac TNFR2 receptors in the TNFR1/
mice mediate the induction of IL-6 after LPS challenge, studies
(5, 28) in other tissue and cell types failed to find a
significant role for TNFR2 in the production of IL-6 elicited by
TNF-.
Further evidence suggests that LPS stimulates cardiac expression of
IL-6 and MCP-1 in a mechanism different from that induced directly by
TNF-. For example, transgenic mice (TNF1.6) that overexpress TNF-
in the myocardium (29) demonstrated elevated MCP-1
expression with no detectable expression of IL-6. Additionally, treatment of TNF1.6 mice with AdTNFR1 completely abrogated the myocardial expression of MCP-1. Therefore, it would appear that LPS
augments the myocardial expression of IL-6 and MCP-1 independently of
or synergistically with the expression of TNF-. Thus, while the
cardiac inflammation associated with myocarditis may be dependent on
TNF- signals mediated through TNFR1 (2), the cardiac
toxicity arising from LPS challenge may be at least partially
independent of TNF--mediated pathways. This finding might explain
the inability of anti-TNF therapies to reverse the cardiotoxicity
associated with endotoxemia because the myocardial expression of
endotoxin-responsive cytokines (i.e., IL-6 and MCP-1) would be unabated
and that of IL-1 only partially reduced.
The mechanisms by which LPS may induce cytokine expression in the
various cells of the heart may include at least two pathways: one CD14
dependent and the other CD14 independent. CD14, a glycosyl-phosphatidyl inositol-anchored glycoprotein expressed in monocytes/macrophages and
neutrophils, binds to the LPS/LPS-binding protein (LBP) complex in
serum to activate macrophages (58).Cardiac myocytes also express CD14 (10, 11). In addition, cells that do not
express membrane-bound CD14 can still respond to the LPS/LBP complex by interacting with soluble CD14 (20). In the present study,
immunohistochemical staining of IL-6 revealed that both myocytes and
infiltrating inflammatory (+CD45) cells were the source of LPS-induced
IL-6 in the mouse heart. This finding is consistent with other reports (22, 56) stating that myocytes, leukocytes, and
endothelial cells all produce IL-6 on LPS challenge. However, this does
not prove that this occurs through a CD14-mediated pathway. Indeed, the
presence of a CD14-independent LPS signaling pathway has been proposed
in various cell types including cardiac myocytes (11). The
CD14-independent pathway seems to be evident only when higher doses of
LPS are administered. In the present study, however, two different
doses of LPS injection resulted in the same cytokine expression profile
in TNFR1-deficient mice. Taken together, these studies suggest that
LPS-induced cardiac production of IL-6 is dependent on CD14 but not
completely dependent on TNF-. Interestingly, in decidual cells, a
similar pattern of CD14-dependent LPS-induced cytokine production was
observed (1); anti-TNF therapy significantly inhibited
IL-1 but not IL-6 production in response to LPS stimulation.
While LPS itself may have direct cardiotoxic effects independent of stimulation of cytokine production (41), IL-6 and MCP-1 are also clearly capable of contributing to cardiac dysfunction. IL-6 is induced in the plasma of patients with endotoxin shock (17, 54) and congestive heart failure (34) and is elevated in the myocardium in animal models of myocarditis or heart failure (46). Furthermore, IL-6 can directly diminish the contractile properties of isolated cardiomyocytes and cardiac tissues (15). In a less direct fashion, because MCP-1 is a prominent signal for the accumulation of monocytes (44), it may be a key mediator in the pathogenesis of myocardial inflammation leading to muscle dysfunction (25).
These studies do suggest that the LPS-induced level of IL-1 in the
plasma and myocardium can be reduced by an anti-TNF- therapy. This
observation is consistent with a report (52) showing that
injection of rodents with TNF- can elicit a modest expression of
IL-1 in some organs, and it would be reasonable that TNF- may act
synergistically with LPS to stimulate IL-1 expression. However, even
though anti-TNF- therapy reduced the LPS-induced serum level of
IL-1 by almost 50% (to 13 ng/ml serum), these cytokine levels are
still well above the concentration range capable of inducing
alterations in gene expression and function in cultured cardiomyocytes
(7, 37, 48). Thus the functional consequence of a partial
reduction in IL-1 expression remains unclear.
In addition to the LPS-induced cytokines recognized for their direct
adverse effects on the myocardium (IL-1, IL-6, and TNF-), IL-10
and IL-12 are also regulated by LPS exposure and play a prominent role
in endotoxemia. IL-10 has been shown to be protective in murine
endotoxemia (21, 47), whereas IL-12 plays an essential role in lethality in endotoxic mice (59). Although their
expression levels were low, both cytokines were significantly induced
by LPS challenge. Furthermore, AdTNFR1 treatment significantly
increased the LPS-induced IL-10 protein levels in both the plasma and
myocardium, consistent with previous findings that anti-TNF therapy
accelerated the early peak but attenuated the delayed peak of IL-10
production induced by LPS (4). However, the cardiac levels
of IL-10 transcripts were not augmented by blocking TNF-
bioactivity, suggesting a complex role for TNF-, perhaps at the
translational or protein processing level, in regulating IL-10
production. On the other hand, AdTNFR1 did not change LPS-induced IL-12
expression in cardiac tissue but markedly reduced the IL-12 plasma
level. While a suppression of systemic IL-12 level may reduce the
severity of endotoxemia, the direct cardiac effects of altering cardiac
levels of either IL-10 or IL-12 remain unknown.
TNF- protein was elevated by LPS injection and further increased
after the treatment with AdTNFR1. This phenomenon, also observed in our
previous study (29) with TNF1.6 mice treated with AdTNFR1,
may arise from several different mechanisms. First, a negative feedback
mechanism present in the translation of TNF- may have been uncoupled
by anti-TNF treatment. Second, although TNF- loses its bioactivity
when bound by soluble TNFR1, it may also gain stability
(39). Because ELISA measures both receptor-bound and free
TNF-, the major contribution to the increase might be due to soluble
receptor-bound TNF-. Whatever the mechanisms of increased TNF-
antigenicity in the mice treated with AdTNFR1, the biological effects
of TNF- were systemically attenuated by the treatment, as shown by
the abrogation of expression of downstream cytokines in plasma.
In summary, anti-TNF treatment differentially altered the expression of
key pro- and anti-inflammatory cytokines (IL-1, IL-6, IL-10, IL-12,
and MCP-1) in the plasma and myocardium during endotoxemia. Although we
did not elucidate the entire mechanism of effects of blocking one
inflammatory component in complex interactions within the cytokine
network, the inability to block myocardial expression of IL-6, IL-12,
and MCP-1 suggests a possible mechanism for the failure of anti-TNF
therapies in the treatment of endotoxin shock syndrome.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. M. Feldman, Cardiovascular Institute of the UPMC Health System, 200 Lothrop S., S 572 Scaife Hall, Pittsburgh, PA 15213 (E-mail: feldmanam{at}msx.upmc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 July 2000; accepted in final form 10 January 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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