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Am J Physiol Heart Circ Physiol 290: H800-H806, 2006. First published September 19, 2005; doi:10.1152/ajpheart.00701.2005
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Cellular and molecular mechanisms underlying LPS-associated myocyte impairment

Samantha A. Tavener and Paul Kubes

Department of Physiology and Biophysics, Immunology Research Group, University of Calgary Medical Centre, Calgary, Alberta, Canada

Submitted 28 June 2005 ; accepted in final form 9 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recently we reported that Toll-like receptor 4 (TLR4)-positive immune cells of unknown identity were responsible for the LPS-induced depression of cardiac myocyte shortening. The aim of this study is to identify the TLR4-positive cell type that is responsible for the LPS-induced cardiac dysfunction. Neither neutrophil depletion alone nor mast cell deficiency had any impact on the impairment of myocyte shortening during LPS treatment. In contrast, LPS-treated, macrophage-deficient mice demonstrated a partial reduction in shortening compared with saline-treated, macrophage-deficient mice. Because the removal of macrophages could only partially restore myocyte shortening, we also investigated the effects of removing both neutrophils and macrophages on myocyte shortening. Interestingly, endotoxemic, neutrophil-depleted, and macrophage-deficient mice had completely restored myocyte shortening. Because both macrophages and neutrophils can produce nitric oxide (NO) and TNF-{alpha}, we examined LPS-treated inducible NO synthase knockout (iNOSKO) mice and TNF receptor (TNFR)-deficient mice. Eliminating both TNFR1 and TNFR2 was required to restore myocyte shortening during LPS treatment, whereas iNOS deficiency had no effect. These data suggest that macrophages and to a lesser degree neutrophils cause cardiac impairment, presumably via TNF-{alpha}.

neutrophils; mast cells; macrophages; endotoxemia; tumor necrosis factor-{alpha}

LPS IS A CELL MEMBRANE COMPONENT shed from gram negative bacteria and is a major contributor to the development of sepsis (9), primarily via Toll-like receptor 4 (TLR4). To date, most studies have examined cardiac responses after animals were treated with LPS with clear evidence that LPS has myocardial depressive properties (15, 34, 41). Recently, we uncovered that myocytes per se are not the direct target of LPS-induced responses observed in the heart over the first few hours of endotoxemia. Rather, TLR4-positive leukocytes are responsible for LPS-induced myocyte dysfunction (52). This was done by making chimeric mice where either the bone marrow cells or myocytes expressed TLR4. Only mice with TLR4 on immune cells demonstrated cardiac impairment in response to LPS. However, the identity of the bone marrow-derived cells was not identified. In the heart there are a number of infiltrating and resident cells, and those immune cells have been shown to express TLR4: neutrophils (28, 33, 43), mast cells (18, 29, 50), and macrophages (28, 33).

Neutrophils or other circulating leukocytes (monocytes) may be responsible for LPS-induced myocyte dysfunction because they are known to rapidly infiltrate cardiac tissue during inflammation. Accumulation of neutrophils in the endotoxemic heart has been reported (13, 41). In fact, a study (15) using Langendorff perfused hearts demonstrated that hearts perfused with leukocyte-depleted, endotoxemic blood had no reduction in pressure generation compared with perfusion with normal endotoxemic blood. In addition, blocking general leukocyte recruitment or leukocyte activation prevented LPS-induced cardiac dysfunction (41, 58), although which leukocytes were responsible was unclear. In vitro studies illustrate that activated neutrophils adhere to myocytes (39, 48) and increase oxidative and nitrosative stress within the cardiac myocyte, ultimately resulting in myocyte dysfunction (11, 39). These studies suggest, albeit indirectly, that neutrophils and/or other circulating leukocytes may be involved in sepsis-associated cardiac dysfunction.

Despite mast cells being involved in inflammation via the production of various mediators, there is no evidence that these cells contribute to LPS-induced myocyte dysfunction. An in vitro study (16) demonstrated that culturing myocytes with mast cell granules can elevate cardiomyocyte apoptosis, suggesting mast cells can certainly cause myocyte damage. During ischemia-reperfusion injury, mast cells (and the release of TNF-{alpha}) were suggested to be key for the recruitment of damaging neutrophils and thus the development of myocardial infarct (12). In addition, the stabilization of mast cells with lodoxamide reduced infarct size during ischemia-reperfusion injury (19). During sepsis, the removal of dipeptidyl peptidase I from mast cells was shown to increase survival (26). Clearly, mast cells can release injurious molecules during infection or ischemia-reperfusion to induce damaging processes.

There are a few studies describing a role for the monocyte/macrophage lineage to reduce cardiac myocyte shortening. One in vitro study (2) illustrated that myocytes treated with supernatant from activated macrophages could reduce shortening. Another in vitro study (46) demonstrated that the adhesion of monocytes via ICAM-1 on cardiac myocytes was shown to reduce shortening. To date, a role for macrophages in LPS-induced cardiac dysfunction per se has not been tested. However, with the availability of macrophage-deficient mice [colony stimulating factor 1 (CSF-1) deficient], this is now possible.

Macrophages, mast cells, neutrophils (1, 3, 37), as well as cardiac myocytes (8, 21, 59), can all release many mediators to affect heart function. TNF-{alpha} has been one of the more avidly studied molecules in endotoxemia and sepsis. In vitro studies showed the treatment of myocytes with TNF-{alpha} alone can reduce myocyte shortening (23, 24). In addition, infusion of TNF-{alpha} showed depression of left ventricular function and myocyte shortening (6). The importance of TNF-{alpha} becomes even more evident with anti-TNF therapy demonstrating a decrease in myocardial infarct size and an effective reduction in mortality in septic animal studies (7). Furthermore, inhibition of TNF-{alpha} with TNF-binding proteins restored the dysfunctional left ventricular developed pressure (LVDP) in endotoxemic rats (31). Although cardiac myocytes express both TNF receptors TNFR1 and TNFR2 (53), it remains unclear which receptor is important.

This study identifies the specific cell type (primarily the monocyte/macrophage lineage) as responsible for LPS-induced myocyte dysfunction. This occurred due to TNF-{alpha}, via both TNFR1 and TNFR2.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and experimental protocol. Male C57BL/6 mice were purchased from Charles River (Montreal, Quebec, Canada), and male mast cell-deficient (WBB6F1/J-kitW/J-kitW-v), macrophage-deficient (B6C3Fe a/a-Csf1op), iNOS-knockout (C57Bl6X129Sv/Ev), TNFR1 (B6,129-Tnfrsf1atm1Mak/J), TNFR2 (B6.129s7-Tnfrsf1btm1Mwm/J), and TNFR1/2-deficient (B6.129-Tnfrsf1atm1Imx/J Tnfrsf1btm1Imx/J) mice and their respective control littermates were purchased from Jackson (Bar Harbor, ME). No differences in the proportion of myeloid or other circulatory leukocytes were detected in the TNFR-deficient mice relative to wild-type controls (32, 42), and the mast cell-deficient mice had no mast cells detected in various organs, including the heart (22). All 152 mice were maintained in a pathogen-free facility until 6–10 wk old, at which time the mice were used. Mice received LPS (10 mg/kg, Escherichia coli serotype 0127:B8) intraperitoneally to induce endotoxemia, a dose demonstrated to ensure cardiac dysfunction without any mortality (39). After 4 h, mice were anethesitized and hearts excised for myocyte isolation. All experimental protocols were reviewed and approved by the University of Calgary Animal Resource Center and conform to the guidelines established by the Canadian Council for Animal Care.

Murine ventricular myocyte isolation. Ventricular myocytes were isolated as previously described (39), and unless otherwise stated, all chemicals were purchased from Sigma-Aldrich. Briefly, 6- to 16-wk-old, male, septic (LPS, E. coli serotype 0127:B8, 10 mg/kg ip, 4 h), wild-type (C57BL/6), neutrophil-depleted, mast cell-deficient or macrophage-deficient mice were anesthetized with methoxyflurane (Metafane; Janssen Pharmaceuticals). Mice were then cervically dislocated, and hearts were removed and placed in ice-cold Tyrode buffer containing (in mmol/l) 139.9 NaCl, 5.4 KCl (BDH), 1.0 Na2HPO4 (BDH), 5.0 HEPES, 10.0 glucose, and 1.0 MgCl2, containing 1 CaCl2. Hearts were then cannulated via the aorta for retrograde perfusion of the coronary arteries with Tyrode buffer containing 1 mmol/l CaCl2 at 2 ml/min for 5 min at 37°C and subsequently perfused with buffer containing no CaCl2 for 5 min. Final perfusion buffer contained 40 µmol/l CaCl2, 20 µg/ml collagenase (Yakult Pharmaceuticals), and 4 µg/ml protease (type XIV bacterial) for 8–10 min. Ventricles were then minced and placed in Tyrode buffer containing 500 µg/ml collagenase, 100 µg/ml protease, and 2.5% bovine serum albumin and placed in a shaking water bath for 10–20 min to obtain a suspension of individual myocytes. Myocytes were placed in DMEM (GIBCO) and were used within 4 h.

Cell shortening. The mechanical properties of the myocytes were measured by using a video-based, edge-detection system (IonOptix, Milton, MA), as previously described (52). Isolated myocytes were allowed to adhere to a glass coverslip in a Warner chamber that was mounted on the stage of an inverted microscope. Myocytes were perfused with 1 ml/min Tyrode buffer and field stimulated at 1 Hz using a threshold voltage of +10%. Shortening and relenghtening were recorded with the soft-edge software (IonOptix). Nonspontaneously contracting myocytes were selected randomly but had to respond to electrical stimulation and have increased shortening in response to a {beta}-adrenergic agonist isoproterenol (0.1 µmol/l, Sigma). It is our experience that myocytes that do not meet these criteria usually die during the protocol, making the data acquisition impossible (52). Therefore, we selected the healthiest myocytes from both the untreated and LPS-treated groups.

Neutrophil depletion. C57BL/6 (wild-type) and macrophage-deficient and their respective control mice received anti-neutrophil antibody (RB6–8C5) intraperitoneally at 150 µg/mouse for 24 h before experimentation, as we have previously described (5, 17) and as per manufacturer's instructions. This treatment removed >90% of the neutrophils from each strain of mice and did not significantly alter the blood levels of other leukocyte populations. In addition, RB6–8C5 treatment has been shown to substantially reduce cardiac neutrophil accumulation (14).

Statistical analysis. Statistical significance between data was assessed using a t-test with a Bonferroni correction for multiple comparisons. Statistical significance was set at P < 0.05.


   RESULTS
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 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Neutrophils are not required for LPS-induced myocyte dysfunction. Neutrophil-depleted mice were generated by using a 24-h anti-neutrophil antibody treatment (Becton Dickinson PharMingen), which was shown to deplete neutrophils by at least 90% in this study and in previous work (5, 17). These mice were also treated with either saline or LPS for 4 h, at which point cardiac myocytes were isolated and cell shortening was measured (Fig. 1). Myocyte shortening from saline-treated, wild-type (5.2 ± 0.7%) and neutrophil-depleted mice (7.1 ± 1.5%) was not significantly different. Cardiac myocytes from LPS-treated, wild-type and neutrophil-depleted mice demonstrated significantly depressed shortening to 2.6 ± 0.3% and 2.5 ± 0.3%, respectively. Despite the elimination of neutrophils, a profound reduction in myocyte shortening remained, illustrating that neutrophils alone are not responsible for the LPS-induced cardiac dysfunction.



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  		Fig. 1. Shortening of ventricular myocytes from saline- and LPS-treated, wild-type (WT) and anti-neutrophil antibody (ANS)-treated mice. *P < 0.05 compared with saline-treated controls. n = minimum number of 4 mice or 11 myocytes/group.

 
Does LPS-induced myocyte impairment involve cardiac mast cells? Another cell type present within the milieu of the endotoxemic heart is the cardiac mast cell. Cardiac myocytes were isolated from saline- and LPS-treated mast cell-deficient mice and littermate controls, and subsequent myocyte shortening was recorded (Fig. 2). The saline-treated, mast cell-deficient and control mice had similar myocyte shortening. Significantly depressed shortening was observed in the LPS-treated controls (2.1 ± 0.2%) and in LPS-treated, mast cell-deficient myocytes (1.4 ± 0.2%). Consequently, the removal of the mast cells does not restore myocyte shortening, suggesting that this cell type is not responsible for LPS-induced myocyte dysfunction.



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  		Fig. 2. Shortening of ventricular myocytes from saline- and LPS-treated, mast cell-control and mast cell-deficient mice. *P < 0.05 compared with saline-treated controls. n = minimum number of 3 mice or 9 myocytes/group.

 
Macrophages are partially involved in myocyte dysfunction associated with LPS treatment. Another candidate that may be responsible for LPS-induced myocyte impairment is the macrophage. Cardiac myocytes were isolated from saline- and LPS-treated macrophage-deficient and littermate control mice, and then myocyte shortening was examined (Fig. 3). Myocytes from the saline-treated, macrophage-control and saline-treated, macrophage-deficient mice had similar shortening. The LPS-treated, macrophage controls demonstrated significantly reduced myocyte shortening of 1.7 ± 0.4%. In contrast, the myocyte shortening of LPS-treated, macrophage-deficient mice was not significantly different from either saline-treated, macrophage-control or -deficient mice.



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  		Fig. 3. Shortening of ventricular myocytes from saline- and LPS-treated, macrophage-control and macrophage-deficient mice. *P < 0.05 compared with saline-treated controls. n = minimum number of 4 mice or 12 myocytes/group.

 
Both macrophages and neutrophils are required for complete LPS-induced myocyte depression. Because the absence of macrophages in the heart during endotoxemia only partially restores myocyte shortening, it is possible that another cell type may be contributing to myocyte depression. Macrophage-deficient mice and littermate controls were treated with anti-neutrophil antibody for 24 h and then with saline or LPS for the last 4 h. The heart was then excised, and the cardiac myocytes were isolated. Saline-treated myocytes from neutrophil-depleted, macrophage-sufficient and -deficient mice demonstrated shortening of 6.2 ± 1.1% and 5.1 ± 1.3%, respectively (Fig. 4). Myocyte shortening from neutrophil-depleted, LPS-treated macrophage control mice was identical to the shortening in the LPS-treated, macrophage-control mice (Fig. 3), further demonstrating no negative effects of neutrophil depletion alone. However, neutrophil depletion in macrophage-deficient mice did not reduce myocyte shortening at all (5.3 ± 1.5%) after LPS treatment. The value is significantly higher than the neutrophil-depleted, LPS-treated, and macrophage-control mice.



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  		Fig. 4. Shortening of ventricular myocytes from saline- and LPS-treated, macrophage-sufficient, ANS-depleted mice and macrophage-deficient, ANS-depleted mice. *P < 0.05 compared with saline-treated controls. {dagger}P < 0.05 compared with LPS-treated, neutrophil-depleted, macrophage-deficient mice. n = minimum number of 3 mice or 9 myocytes/group.

 
Could the production of mediators, such as nitric oxide, be responsible for LPS-associated myocyte impairment? Macrophages and neutrophils will generate reactive nitrogen species, like nitric oxide (NO), which may react with other molecules and be damaging to both invading organisms and resident tissue. Cardiac myocytes from saline and LPS-treated inducible NO synthase (iNOS) knockout (iNOSKO) mice were isolated, and shortening was analyzed (Fig. 5). Myocyte shortening from the saline-treated, wild-type and iNOSKO mice were identical, whereas myocytes from LPS-treated, wild-type and iNOSKO mice demonstrated significantly depressed shortening of 2.6 ± 0.3% and 2.3 ± 0.3%, respectively. Thus NO may not be responsible for depressed myocyte shortening associated with LPS treatment in our model.



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  		Fig. 5. Shortening of ventricular myocytes from saline- and LPS-treated WT and inducible nitric oxide synthase knockout (iNOSKO) mice. *P < 0.05 compared with saline-treated controls. n = minimum number of 3 mice or 11 myocytes/group.

 
TNF-{alpha} can cause myocyte dysfunction. Although the literature suggests that TNF-{alpha} in vitro can cause myocyte depression, the effects of TNF-{alpha} on myocytes during endotoxemia are unclear. Additionally, the importance of TNFR1 and TNFR2 signaling for myocyte dysfunction are also unresolved. Cardiac myocytes were isolated from saline- and LPS-treated TNFR1-deficient and TNFR2-deficient mice, and myocyte shortening was examined (Fig. 6). Similar myocyte shortening was demonstrated for all saline-treated groups. Furthermore, all groups showed equally depressed myocyte shortening with LPS treatment. Because both receptors have been reported to be involved in various TNF-{alpha}-induced effects, cardiac myocytes from double-deficient (TNFR1/2–/–) mice were isolated, and shortening was examined (Fig. 7). Myocytes from the saline-treated, wild-type and TNFR1/2–/– mice demonstrated shortening of 5.1 ± 0.7% and 6.8 ± 0.8%, respectively, whereas myocytes from the LPS-treated, wild-type mice have depressed shortening of 2.6 ± 0.3%. Myocyte shortening from the LPS-treated, TNFR1/2–/– mice had comparable shortening with the saline-treated group (5.1 ± 0.7%), suggesting that TNF-{alpha} is required for LPS-induced myocyte impairment.



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  		Fig. 6. Shortening of ventricular myocytes from saline- and LPS-treated WT, TNF receptor (TNFR)1, and TNFR2 deficient mice. *P < 0.05 compared with saline-treated controls. n = minimum number of 4 mice or 9 myocytes/group.

 


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  		Fig. 7. Shortening of ventricular myocytes from saline- and LPS-treated WT and TNFR double deficient (TNFR1/2–/–) mice. *P < 0.05 compared with saline-treated controls. n = minimum number of 5 mice or 10 myocytes/group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Recently, we reported a role for TLR4 positive immune cells rather than parenchymal cells (myocytes or fibroblasts) in LPS-induced myocyte dysfunction (52). Yet, the TLR4-positive immune cell type that was responsible for myocyte impairment during endotoxemia was not addressed. Our results herein show that neither neutrophils nor mast cells alone are central in the depression of myocyte shortening during LPS treatment. In contrast, macrophages appear to play a significant role. Interestingly, LPS-treated, neutrophil-depleted, and macrophage-deficient mice had completely restored myocyte shortening. Although macrophages and neutrophils can both produce NO (4, 24) and TNF-{alpha} (37, 54), the elimination of both TNFR1 and TNFR2 was required to restore myocyte shortening during LPS treatment, whereas removal of only one TNFR or iNOS had no effect on the depressed myocyte shortening.

Although mast cells are not often considered to be important immune cells in endotoxemia or septicemia, they have been reported to be the primary source of TNF-{alpha} in the first few hours of infection in the murine peritoneal cavity (10, 25). Therefore, we hypothesized that mast cells known to reside in the heart (20, 49) and to harbor cytokines (12) were the cells involved in myocyte dysfunction. We made use of the well-established mouse system, the W/Wv mice lacking c-kit, a critical receptor for the mast cell growth factor. These mice lack mast cells in essentially all tissues, including the heart (35). Despite this deficiency, we observed absolutely no improvement in the impaired myocyte shortening associated with LPS.

Unlike studies with general leukocyte-depletion protocols, there is little direct evidence that specific removal of neutrophils can alleviate cardiac depression during endotoxemia. In fact, neither vinblastine nor anti-neutrophil antibody treatment had any effect on the reduction in LVDP induced by LPS (40). This group also showed that blocking ICAM-1 in endotoxemic mice improved LVDP but did not block neutrophil accumulation. In agreement, another study (27) using nonspecific neutrophil depletion (cyclophosphamide) was unable to show a reduction in cardiac damage during E. coli-induced multiple organ failure in guinea pigs. Therefore, improved cardiac function was suggested to be independent of neutrophil accumulation. These studies support our data that show LPS-treated, neutrophil-depleted mice still have impaired myocyte function.

Our data reveal that monocytes and macrophages contribute to myocyte impairment during endotoxemia. Interestingly, not all the myocyte dysfunction was alleviated, but the macrophage-deficient mice did have a small percentage of tissue macrophages remaining (57). However, these macrophages were nonfunctional [impaired ability to release TNF-{alpha} (56)] and thus would be unlikely to cause LPS-associated myocyte dysfunction. Yet another possibility to explain the lack of complete inhibition of cardiac impairment is that neutrophils could contribute to the remaining myocyte depression during LPS treatment. Indeed, the removal of neutrophils in macrophage-deficient mice during endotoxemia exhibited normal myocyte shortening. Although neutrophil depletion alone could not alleviate any of the myocyte dysfunction, this may be due to the overwhelming arsenal of cytokines (e.g., TNF-{alpha}) produced by macrophages that masks the more subtle impairment induced by neutrophils per se. Indeed, neutrophils can also produce molecules like TNF-{alpha} but in barely detectable quantities. This would suggest that primarily macrophages but also neutrophils are required for LPS-induced myocyte dysfunction. Nevertheless, one cannot discount a role for neutrophils in this model of myocardial depression.

There have been a number of studies looking at the effects of NO on cardiac performance. In fact, Poon et al. (39) showed that extravascular neutrophils containing iNOS can adhere to myocytes and cause a rapid decrease in myocyte shortening, whereas neutrophils from iNOS-deficient mice were unable to cause myocyte damage. An important difference between the Poon et al. study and this study is the direct application of maximally activated neutrophils to myocytes in vitro in the former study. Clearly, under these conditions, neutrophils can produce sufficient NO to induce myocyte dysfunction. In contrast, in vivo, the neutrophils either were not sufficiently activated and/or did not have the same degree of access to myocytes as in vitro. Alternatively, macrophage iNOS may be a more relevant source, because macrophages in this study were required for myocyte dysfunction during endotoxemia. However, in our system, no benefit was observed in myocyte shortening from the endotoxemic iNOS-deficient mice, suggesting that despite the fact that neutrophils and macrophages have both been reported to have the capacity to upregulate iNOS to cause myocyte dysfunction, this was not a major mechanism in our model. This suggests that iNOS is not required in endotoxemic cardiac dysfunction, which agrees with other studies. Aminoguanidine or S-methylisothioureasulfate (inhibitors of iNOS) did not improve LVDP of LPS-treated rats (30, 55). Collectively, this work suggests that iNOS plays a minimal role during the early stages of endotoxemia.

Our data suggest that TNF-{alpha} is the key mediator involved in the first 4 h of LPS-induced myocyte dysfunction. Both divergent and redundant roles for TNFR1 and TNFR2 have been suggested in some disease models. A study using TNFR1-deficient mice (36) or TNFR1-blocking monoclonal antibody (45) has demonstrated protection from lethal shock with the use of LPS and galactosamine. In contrast, an antibody that blocks TNFR2 did not protect mice from lethal shock. Our data suggest that both TNFR1 and TNFR2 are involved in LPS-induced myocyte dysfunction, consistent with the observation that both TNFR1- and TNFR2-deficient mice are sensitive to LPS-induced mortality (36). In fact, both TNFR1 and TNFR2 have been reported as necessary in other biological responses, including TNF-mediated endothelial activation (44), expression of adhesion molecules (47), and TNF-induced cytotoxicity of tumor cells (44). Two different models, ligand passing and TNFR heterocomplexing, have been suggested to explain the cooperation between TNFR1 and TNFR2. The ligand passing model suggests that high-affinity TNFR2 (10- to 20-fold higher affinity than TNFR1) can rapidly associate and dissociate with TNF-{alpha}, thereby allowing a TNF-{alpha} association with TNFR1 (51). In contrast, the heterocomplexing model suggests that adjacent TNFR1 and TNFR2 can share downstream-signaling molecules (38), such that there exists a functional overlap.

In conclusion, macrophages appear to play an important role in LPS-induced myocyte dysfunction with a more minor contribution from infiltrating neutrophils. Although both macrophages and neutrophils can produce NO and TNF-{alpha}, we could demonstrate a role only for TNF-{alpha} in LPS-induced myocyte dysfunction in this model system. Intriguingly, the elimination of both TNFR1 and TNFR2 was necessary to restore myocyte shortening during LPS treatment. Therefore, both TNFR1 and TNFR2 are essential for myocyte dysfunction during endotoxemia.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a Canadian Institute of Health Operating Grant and Group Grant.


   ACKNOWLEDGMENTS
 
P. Kubes is an Alberta Heritage Foundation for Medical Research Scientist and Canada Research Chair.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Kubes, Dept. of Physiology and Biophysics, Institute of Infection, Immunity and Inflammation Univ. of Calgary, 3330 Hospital Dr. NW, Calgary, AB T2N 4N1, Canada (e- mail: pkubes{at}ucalgary.ca)

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.


   REFERENCES
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  1. Asehnoune K, Strassheim D, Mitra S, Kim JY, and Abraham E. Involvement of reactive oxygen species in Toll-like receptor 4-dependent activation of NF-kappa B. J Immunol 172: 2522–2529, 2004.[Abstract/Free Full Text]
  2. Balligand JL, Ungureanu D, Kelly RA, Kobzik L, Pimental D, Michel T, and Smith TW. Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium. J Clin Invest 91: 2314–2319, 1993.[ISI][Medline]
  3. Bazzoni F, Cassatella MA, Laudanna C, and Rossi F. Phagocytosis of opsonized yeast induces tumor necrosis factor-alpha mRNA accumulation and protein release by human polymorphonuclear leukocytes. J Leukoc Biol 50: 223–228, 1991.[Abstract]
  4. Benjamim CF, Ferreira SH, and Cunha FQ. Role of nitric oxide in the failure of neutrophil migration in sepsis. J Infect Dis 182: 214–223, 2000.[CrossRef][ISI][Medline]
  5. Bonder CS, Ajuebor MN, Zbytnuik LD, Kubes P, and Swain MG. Essential role for neutrophil recruitment to the liver in concanavalin A-induced hepatitis. J Immunol 172: 45–53, 2004.[Abstract/Free Full Text]
  6. Bozkurt B, Kribbs SB, Clubb FJ Jr, Michael LH, Didenko VV, Hornsby PJ, Seta Y, Oral H, Spinale FG, and Mann DL. Pathophysiologically relevant concentrations of tumor necrosis factor-alpha promote progressive left ventricular dysfunction and remodeling in rats. Circulation 97: 1382–1391, 1998.[Abstract/Free Full Text]
  7. Cohen J and Exley AR. Treatment of septic shock with antibodies to tumour necrosis factor. Schweiz Med Wochenschr 123: 492–496, 1993.[ISI][Medline]
  8. Comstock KL, Krown KA, Page MT, Martin D, Ho P, Pedraza M, Castro EN, Nakajima N, Glembotski CC, Quintana PJ, and Sabbadini RA. LPS-induced TNF-alpha release from and apoptosis in rat cardiomyocytes: obligatory role for CD14 in mediating the LPS response. J Mol Cell Cardiol 30: 2761–2775, 1998.[CrossRef][ISI][Medline]
  9. Dunn DL, Bogard WC Jr, and Cerra FB. Enhanced survival during murine gram-negative bacterial sepsis by use of a murine monoclonal antibody. Arch Surg 120: 50–53, 1985.[Abstract]
  10. Echtenacher B, Mannel DN, and Hultner L. Critical protective role of mast cells in a model of acute septic peritonitis. Nature 381: 75–77, 1996.[CrossRef][Medline]
  11. Entman ML, Youker K, Shappell SB, Siegel C, Rothlein R, Dreyer WJ, Schmalstieg FC, and Smith CW. Neutrophil adherence to isolated adult canine myocytes. Evidence for a CD18-dependent mechanism. J Clin Invest 85: 1497–1506, 1990.[ISI][Medline]
  12. Frangogiannis NG, Lindsey ML, Michael LH, Youker KA, Bressler RB, Mendoza LH, Spengler RN, Smith CW, and Entman ML. Resident cardiac mast cells degranulate and release preformed TNF-alpha, initiating the cytokine cascade in experimental canine myocardial ischemia/reperfusion. Circulation 98: 699–710, 1998.[Abstract/Free Full Text]
  13. Goddard CM, Poon BY, Klut ME, Wiggs BR, vanEeden SF, Hogg JC, and Walley KR. Leukocyte activation does not mediate myocardial leukocyte retention during endotoxemia in rabbits. Am J Physiol Heart Circ Physiol 275: H1548–H1557, 1998.[Abstract/Free Full Text]
  14. Grabie N, Hsieh DT, Buono C, Westrich JR, Allen JA, Pang H, Stavrakis G, and Lichtman AH. Neutrophils sustain pathogenic CD8+ T cell responses in the heart. Am J Pathol 163: 2413–2420, 2003.[Abstract/Free Full Text]
  15. Granton JT, Goddard CM, Allard MF, van Eeden S, and Walley KR. Leukocytes and decreased left-ventricular contractility during endotoxemia in rabbits. Am J Respir Crit Care Med 155: 1977–1983, 1997.[Abstract]
  16. Hara M, Matsumori A, Ono K, Kido H, Hwang MW, Miyamoto T, Iwasaki A, Okada M, Nakatani K, and Sasayama S. Mast cells cause apoptosis of cardiomyocytes and proliferation of other intramyocardial cells in vitro. Circulation 100: 1443–1449, 1999.[Abstract/Free Full Text]
  17. Hwang JM, Yamanouchi J, Santamaria P, and Kubes P. A critical temporal window for selectin-dependent CD4+ lymphocyte homing and initiation of late-phase inflammation in contact sensitivity. J Exp Med 199: 1223–1234, 2004.[Abstract/Free Full Text]
  18. Ikeda T and Funaba M. Altered function of murine mast cells in response to lipopolysaccharide and peptidoglycan. Immunol Lett 88: 21–26, 2003.[CrossRef][ISI][Medline]
  19. Jolly SR, Abrams GD, Romson JL, Bailie MB, and Lucchesi BR. Effects of lodoxamide on ischemic reperfused myocardium. J Cardiovasc Pharmacol 4: 441–448, 1982.[ISI][Medline]
  20. Joseph J, Kennedy RH, Devi S, Wang J, Joseph L, and Hauer-Jensen M. Protective role of mast cells in homocysteine-induced cardiac remodeling. Am J Physiol Heart Circ Physiol 288: H2541–H2645, 2005.[Abstract/Free Full Text]
  21. Kapadia S, Lee J, Torre-Amione G, Birdsall HH, Ma TS, and Mann DL. Tumor necrosis factor-alpha gene and protein expression in adult feline myocardium after endotoxin administration. J Clin Invest 96: 1042–1052, 1995.[ISI][Medline]
  22. Kitamura Y, Go S, and Hatanaka K. Decrease of mast cells in W/Wv mice and their increase by bone marrow transplantation. Blood 52: 447–452, 1978.[Abstract/Free Full Text]
  23. Kumar A, Brar R, Wang P, Dee L, Skorupa G, Khadour F, Schulz R, and Parrillo JE. Role of nitric oxide and cGMP in human septic serum-induced depression of cardiac myocyte contractility. Am J Physiol Regul Integr Comp Physiol 276: R265–R276, 1999.[Abstract/Free Full Text]
  24. Kumar A, Thota V, Dee L, Olson J, Uretz E, and Parrillo JE. Tumor necrosis factor alpha and interleukin 1beta are responsible for in vitro myocardial cell depression induced by human septic shock serum. J Exp Med 183: 949–958, 1996.[Abstract/Free Full Text]
  25. Malaviya R, Ikeda T, Ross E, and Abraham SN. Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-alpha. Nature 381: 77–80, 1996.[CrossRef][Medline]
  26. Mallen-St Clair J, Pham CT, Villalta SA, Caughey GH, and Wolters PJ. Mast cell dipeptidyl peptidase I mediates survival from sepsis. J Clin Invest 113: 628–634, 2004.[CrossRef][ISI][Medline]
  27. Mallick AA, Ishizaka A, Stephens KE, Hatherill JR, Tazelaar HD, and Raffin TA. Multiple organ damage caused by tumor necrosis factor and prevented by prior neutrophil depletion. Chest 95: 1114–1120, 1989.[Abstract/Free Full Text]
  28. Marsik C, Mayr F, Cardona F, Derhaschnig U, Wagner OF, and Jilma B. Endotoxaemia modulates Toll-like receptors on leucocytes in humans. Br J Haematol 121: 653–656, 2003.[CrossRef][ISI][Medline]
  29. McCurdy JD, Lin TJ, and Marshall JS. Toll-like receptor 4-mediated activation of murine mast cells. J Leukoc Biol 70: 977–984, 2001.[Abstract/Free Full Text]
  30. Meng X, Ao L, Brown JM, Fullerton DA, Banerjee A, and Harken AH. Nitric oxide synthase is not involved in cardiac contractile dysfunction in a rat model of endotoxemia without shock. Shock 7: 111–118, 1997.[ISI][Medline]
  31. Meng X, Ao L, Meldrum DR, Cain BS, Shames BD, Selzman CH, Banerjee A, and Harken AH. TNF-{alpha} and myocardial depression in endotoxemic rats: temporal discordance of an obligatory relationship. Am J Physiol Regul Integr Comp Physiol 275: R502–R508, 1998.[Abstract/Free Full Text]
  32. Mizgerd JP, Peschon JJ, and Doerschuk CM. Roles of tumor necrosis factor receptor signaling during murine Escherichia coli pneumonia. Am J Respir Cell Mol Biol 22: 85–91, 2000.[Abstract/Free Full Text]
  33. Muzio M, Bosisio D, Polentarutti N, D'amico G, Stoppacciaro A, Mancinelli R, van't Veer C, Penton-Rol G, Ruco LP, Allavena P, and Mantovani A. Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells. J Immunol 164: 5998–6004, 2000.[Abstract/Free Full Text]
  34. Natanson C, Danner RL, Elin RJ, Hosseini JM, Peart KW, Banks SM, MacVittie TJ, Walker RI, and Parrillo JE. Role of endotoxemia in cardiovascular dysfunction and mortality. Escherichia coli and Staphylococcus aureus challenges in a canine model of human septic shock. J Clin Invest 83: 243–251, 1989.[ISI][Medline]
  35. Niwa Y, Kasugai T, Ohno K, Morimoto M, Yamazaki M, Dohmae K, Nishimune Y, Kondo K, and Kitamura Y. Anemia and mast cell depletion in mutant rats that are homozygous at "white spotting (Ws)" locus. Blood 78: 1936–1941, 1991.[Abstract/Free Full Text]
  36. Peschon JJ, Torrance DS, Stocking KL, Glaccum MB, Otten C, Willis CR, Charrier K, Morrissey PJ, Ware CB, and Mohler KM. TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation. J Immunol 160: 943–952, 1998.[Abstract/Free Full Text]
  37. Petrofsky M and Bermudez LE. Neutrophils from Mycobacterium avium-infected mice produce TNF-alpha, IL-12, and IL-1 beta and have a putative role in early host response. Clin Immunol 91: 354–358, 1999.[CrossRef][ISI][Medline]
  38. Pinckard JK, Sheehan KC, and Schreiber RD. Ligand-induced formation of p55 and p75 tumor necrosis factor receptor heterocomplexes on intact cells. J Biol Chem 272: 10784–10789, 1997.[Abstract/Free Full Text]
  39. Poon BY, Raharjo E, Patel KD, Tavener S, and Kubes P. Complexity of inducible nitric oxide synthase: cellular source determines benefit versus toxicity. Circulation 108: 1107–1112, 2003.[Abstract/Free Full Text]
  40. Raeburn CD, Calkins CM, Zimmerman MA, Song Y, Ao L, Banerjee A, Harken AH, and Meng X. ICAM-1 and VCAM-1 mediate endotoxemic myocardial dysfunction independent of neutrophil accumulation. Am J Physiol Regul Integr Comp Physiol 283: R477–R486, 2002.[Abstract/Free Full Text]
  41. Raeburn CD, Calkins CM, Zimmerman MA, Song Y, Ao L, Banerjee A, Meng X, and Harken AH. Vascular cell adhesion molecule-1 expression is obligatory for endotoxin-induced myocardial neutrophil accumulation and contractile dysfunction. Surgery 130: 319–325, 2001.[CrossRef][ISI][Medline]
  42. Rebel VI, Hartnett S, Hill GR, Lazo-Kallanian SB, Ferrara JL, and Sieff CA. Essential role for the p55 tumor necrosis factor receptor in regulating hematopoiesis at a stem cell level. J Exp Med 190: 1493–1504, 1999.[Abstract/Free Full Text]
  43. Sabroe I, Jones EC, Usher LR, Whyte MK, and Dower SK. Toll-like receptor (TLR)2 and TLR4 in human peripheral blood granulocytes: a critical role for monocytes in leukocyte lipopolysaccharide responses. J Immunol 168: 4701–4710, 2002.[Abstract/Free Full Text]
  44. Shalaby MR, Sundan A, Loetscher H, Brockhaus M, Lesslauer W, and Espevik T. Binding and regulation of cellular functions by monoclonal antibodies against human tumor necrosis factor receptors. J Exp Med 172: 1517–1520, 1990.[Abstract/Free Full Text]
  45. Sheehan KC, Pinckard JK, Arthur CD, Dehner LP, Goeddel DV, and Schreiber RD. Monoclonal antibodies specific for murine p55 and p75 tumor necrosis factor receptors: identification of a novel in vivo role for p75. J Exp Med 181: 607–617, 1995.[Abstract/Free Full Text]
  46. Simms MG and Walley KR. Activated macrophages decrease rat cardiac myocyte contractility: importance of ICAM-1-dependent adhesion. Am J Physiol Heart Circ Physiol 277: H253–H260, 1999.[Abstract/Free Full Text]
  47. Slowik MR, De Luca LG, Fiers W, and Pober JS. Tumor necrosis factor activates human endothelial cells through the p55 tumor necrosis factor receptor but the p75 receptor contributes to activation at low tumor necrosis factor concentration. Am J Pathol 143: 1724–1730, 1993.[Abstract]
  48. Smith CW, Entman ML, Lane CL, Beaudet AL, Ty TI, Youker K, Hawkins HK, and Anderson DC. Adherence of neutrophils to canine cardiac myocytes in vitro is dependent on intercellular adhesion molecule-1. J Clin Invest 88: 1216–1223, 1991.[ISI][Medline]
  49. Sperr WR, Bankl HC, Mundigler G, Klappacher G, Grossschmidt K, Agis H, Simon P, Laufer P, Imhof M, Radaszkiewicz T, Glogar D, Lechner K, and Valent P. The human cardiac mast cell: localization, isolation, phenotype, and functional characterization. Blood 84: 3876–3884, 1994.[Abstract/Free Full Text]
  50. Supajatura V, Ushio H, Nakao A, Akira S, Okumura K, Ra C, and Ogawa H. Differential responses of mast cell Toll-like receptors 2 and 4 in allergy and innate immunity. J Clin Invest 109: 1351–1359, 2002.[CrossRef][ISI][Medline]
  51. Tartaglia LA, Pennica D, and Goeddel DV. Ligand passing: the 75-kDa tumor necrosis factor (TNF) receptor recruits TNF for signaling by the 55-kDa TNF receptor. J Biol Chem 268: 18542–18548, 1993.[Abstract/Free Full Text]
  52. Tavener SA, Long EM, Robbins SM, McRae KM, Van Remmen H, and Kubes P. Immune cell Toll-like receptor 4 is required for cardiac myocyte impairment during endotoxemia. Circ Res 95: 700–707, 2004.[Abstract/Free Full Text]
  53. Torre-Amione G, Kapadia S, Lee J, Bies RD, Lebovitz R, and Mann DL. Expression and functional significance of tumor necrosis factor receptors in human myocardium. Circulation 92: 1487–1493, 1995.[Abstract/Free Full Text]
  54. Vogel S, Hirschfeld MJ, and Perera PY. Signal integration in lipopolysaccharide (LPS)-stimulated murine macrophages. J Endotoxin Res 7: 237–241, 2001.[CrossRef][ISI]
  55. Vona-Davis L, Wearden P, Hill J, and Hill R. Cardiac response to nitric oxide synthase inhibition using aminoguanidine in a rat model of endotoxemia. Shock 17: 404–410, 2002.[CrossRef][ISI][Medline]
  56. Wiktor-Jedrzejczak W, Ansari AA, Szperl M, and Urbanowska E. Distinct in vivo functions of two macrophage subpopulations as evidenced by studies using macrophage-deficient op/op mouse. Eur J Immunol 22: 1951–1954, 1992.[ISI][Medline]
  57. Wiktor-Jedrzejczak WW, Ahmed A, Szczylik C, and Skelly RR. Hematological characterization of congenital osteopetrosis in op/op mouse. Possible mechanism for abnormal macrophage differentiation. J Exp Med 156: 1516–1527, 1982.[Abstract/Free Full Text]
  58. Wolfard A, Kaszaki J, Szabo C, Szalay L, Nagy S, and Boros M. Prevention of early myocardial depression in hyperdynamic endotoxemia in dogs. Shock 13: 46–51, 2000.[ISI][Medline]
  59. Wright G, Singh IS, Hasday JD, Farrance IK, Hall G, Cross AS, and Rogers TB. Endotoxin stress-response in cardiomyocytes: NF-{kappa}B activation and tumor necrosis factor-{alpha} expression. Am J Physiol Heart Circ Physiol 282: H872–H879, 2002.[Abstract/Free Full Text]



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