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(Circulation. 1995;92:1487-1493.)
© 1995 American Heart Association, Inc.

Articles

Expression and Functional Significance of Tumor Necrosis Factor Receptors in Human Myocardium

Guillermo Torre-Amione, MD, PhD; Samir Kapadia, MD; Joseph Lee, MD; Roger D. Bies, MD; Russel Lebovitz, MD, PhD; Douglas L. Mann, MD

From the Cardiology Section (G.T.-A., S.K., J.L., R.L., D.L.M.), Departments of Medicine and Pathology, Veterans Administration Medical Center and Baylor College of Medicine, Houston, Tex; and the Cardiology Section (R.D.B.), Department of Medicine, University of Colorado (Denver).

Correspondence to Douglas L. Mann, MD, Cardiology Section, VA Medical Center, 2002 Holcombe Blvd, Houston, TX 77030.

	Abstract

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Background Tumor necrosis factor– (TNF-), a proinflammatory cytokine with potent negative inotropic properties, is elaborated in septic shock, acute myocarditis, reperfusion injury, and congestive heart failure. TNF- acts by binding to two specific receptors: TNFR1 and TNFR2. However, neither the presence nor the significance of TNF receptors has been studied in the adult mammalian heart.

Methods and Results In the present study, we showed that the adult heart expresses mRNA and receptor proteins for TNFR1 and TNFR2. Moreover, immunohistochemical staining studies localized TNFR1 and TNFR2 to the cardiac myocyte, providing a potential signaling pathway for the deleterious effects of TNF-. The functional significance of the expression of TNFR1 and TNFR2 was explored with the use of a simple cell motion assay in which we assessed the effect(s) of TNF- mutants known to bind selectively to human TNFR1 and TNFR2. We showed that the negative inotropic effect of wild-type TNF- in isolated feline cardiac myocytes was mimicked by the TNF mutant that binds to TNFR1, whereas the TNF mutant that binds to TNFR2 had no significant effect on cell motion.

Conclusions Results of the present study show that the adult human heart expresses both mRNA and receptor proteins for TNFR1 and TNFR2; moreover, the negative inotropic effects of TNF- in adult cardiac myocytes appear to be initiated by activation of TNFR1.

Key Words: tumor necrosis factor • myocardium • myocytes

	Introduction

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Tumor necrosis factor– (TNF-) was originally discovered as a protein that produced necrotizing effects in certain types of transplantable mouse tumors.¹ Subsequent studies have shown that the spectrum of biological activities for TNF- is not limited to cytotoxic effects but rather that TNF- exerts pleiotropic effects in a wide variety of mammalian cell types, including adult cardiac myocytes.^{2 3 4} Although the precise role of TNF- in the heart is not known, the elaboration of TNF- in cardiac pathophysiological contexts in which left ventricular dysfunction develops, such as septic shock, myocarditis, cardiac allograft rejection, and congestive heart failure,^{5 6 7 8 9 10 11} suggests that TNF- may play a pathogenetic role in these disease states. However, the basic cellular and molecular mechanisms responsible for signaling the negative inotropic effects of TNF- are not known.

TNF- initiates its biological effects by binding to two distinct cell surface receptors with approximate molecular masses of 55 kd (TNFR1) and 75 kd (TNFR2). Although both TNF receptor species have been cloned in humans^{12 13 14 15} and are believed to be expressed widely in mammalian cells,^{16 17} it is not known whether they are expressed in the adult heart. Accordingly, the purpose of the present study was to determine whether TNF- receptors are expressed in the adult human heart as well as to determine their functional significance. In the present study, we showed that the human heart expresses both mRNA and receptor proteins for TNFR1 and TNFR2. Moreover, results from the present study suggest that the negative inotropic effects of TNF- are initiated by activation of TNFR1.

	Methods

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Tissue Procurement
Myocardial samples were obtained from organ donors (mean age, 32.2±7.8 years) whose hearts were initially considered for cardiac transplantation but were subsequently deemed unsuitable for transplantation because of blood type or size incompatibility (Table 1). There was no history of primary myocardial disease or evidence of active infection or malignancy in these patients before explantation. All organ donors had normal left function as documented by two-dimensional echocardiography. Moreover, trabeculae isolated from the right ventricles of these subjects demonstrated normal rates of force development and normal levels of peak tension at baseline and after the administration of isoproterenol.^{18 19} Immediately after elective cardiac explantation, tissue aliquots from the still-beating heart were immediately frozen in liquid nitrogen and stored at -80°C until analysis.

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics of Organ Donors

Myocardial TNF Receptors
Myocardial TNF Receptor Gene Expression
Total RNA was extracted from normal myocardium by the guanidinium thiocyanate method.²⁰ Total RNA was denatured at 65°C for 10 minutes, size-fractionated on a 1% agarose gel containing 2.2 mol/L formaldehyde (10 µg/lane), transferred onto nylon membrane (GeneScreen, Dupont NEN), and hybridized sequentially to random primed cDNA probes.²⁰ The following probes were used for Northern blot analysis: a 1.0-kb EcoR1 fragment of the human TNFR1 (a gift from C. Smith, Immunex); a 0.64-kb Not I/Bgl I fragment of the human TNFR2 (a gift from C. Smith, Immunex); and a 0.5-kb Xba I/HindIII fragment of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as an internal control. The membranes were washed once with standard saline citrate and 0.1% sodium dodecyl sulfate at 55°C for 30 minutes, air dried, and exposed to Kodak X-Omat A film at -70°C.

Myocardial TNF Receptor Proteins
Frozen sections of myocardium were cut into 0.5- to 1.0-g pieces, suspended in a phosphate-buffered saline (PBS) solution containing protease inhibitors (1.49 mmol/L phenylmethylsulfonyl fluoride, 475.6 µmol/L leupeptin, and 0.31 µmol/L aprotinin), and homogenized for 30 to 60 seconds with a Polytron PT-3000 (Brinkman Instruments). The myocardial homogenates were centrifuged for 20 minutes at 4°C at 20 000g. The resultant cell pellet was solubilized according to the method of Stauber et al.²¹ Briefly, the cell pellet was resuspended in PBS with proteinase inhibitors and 1% Triton X-100. The pellet was incubated for 1 hour at 4°C, and the solubilized proteins were centrifuged for 20 minutes at 20 000g at 4°C to remove particulate debris. The supernatant, which contained the solubilized cell membrane–bound TNF receptor,²¹ was used for analysis of TNFR1 and TNFR2. The protein content of the membrane fraction was determined with a commercially available assay (BCA, Pierce), with bovine serum albumin as a standard.

Levels of TNFR1 and TNFR2 in the membrane fraction were measured with a "sandwich" ELISA, with commercially available kits used for the detection of human TNFR1 and TNFR2 (Quantikine, R&D Systems). The antibodies used in these immunoassays have been characterized extensively for specificity by the suppliers, are not influenced adversely by the presence of TNF- or TNF-ß, and have a lower limit of detection of 7.8 pg/mL for both receptors. Briefly, 1.3±0.1 mg/mL of solubilized myocardial membrane protein was diluted 1:5 in "calibrator diluent"; 200 µL of unknown sample was then added to each well along with the appropriate recombinant human soluble TNFR1 and TNFR2 standards, and the immunoassay was performed exactly according to the manufacturer's suggestions. After the addition of substrate, results were analyzed spectrophotometrically at a wavelength of 450 nm with a microtiter plate reader. Results were expressed as picomoles per liter of TNF receptor per gram of membrane protein and represent the mean value of two separate measurements performed in duplicate.

Two preliminary control studies were performed to validate the ELISA technique for quantifying membrane-bound proteins. First, to confirm that the homogenizing buffer (used to solubilize the membrane fraction) did not adversely influence the ability of the ELISA to detect solubilized TNFR1 and TNFR2, we diluted purified recombinant TNFR1 (148 pmol/L) and TNFR2 (156 pmol/L) with either homogenizing buffer or "assay diluent"; there was no difference in the immunodetectable levels of TNFR1 or TNFR2 obtained with homogenization buffer compared with the levels obtained with assay diluent. Second, we examined the ability of this method to detect levels of TNFR1 and TNFR2 in two human cell lines known to express high levels of TNFR2: U-937 cells (a histiocytic leukemia cell line) and peripheral blood mononuclear cells. These control studies showed that the ratio of TNFR2 to TNFR1 was 2.6 and 1.8 for U-937 cells and monocytes, respectively, which is similar to that reported in the literature for ligand-binding assays.^{22 23}

Immunolocalization of TNF Receptors
Immunostaining of human myocardial samples for TNFR1 and TNFR2 was performed using a modification of the method described by Yelavarthi and Hunt²⁴ for immunolocalizing TNF receptors in human placenta. Briefly, serial sections of myocardium were incubated with nonimmune rabbit serum or polyclonal rabbit antibody (1:50 dilution) against TNFR1 (provided by C. Smith, Immunex) or polyclonal rabbit antibody (1:100 dilution) against TNFR2 (provided by C. Smith, Immunex). The primary antibodies used have been characterized extensively by the provider and do not cross-react between TNF receptor subtypes. Myocardial sections were incubated for 16 to 18 hours, washed three times in PBS, and then incubated for 45 minutes with a biotinylated anti-rabbit antibody (diluted 1:100). The tissue sections were then washed three times in PBS and developed with a commercially available kit (BioGenex) that uses alkaline phosphatase as the enzyme label and Fast Red (4-chloro-2-methyl-benzene-diasonium chloride naphalene disulfonate) as the chromogen substrate.

Functional Analysis of TNF Receptors
To examine the functional significance of TNF receptors in the cardiac myocytes, we used a simple cell motion assay³ to characterize the effects of two "TNF- mutants." These mutated ligands have double-point mutations in their TNF receptor binding domains,²⁵ such that they have specificity for binding to either human TNFR1 (corresponding mutant TNFM1; provided by W. Lesslauer, F. Hoffman–La Roche²⁵ ) or to TNFR2 (corresponding mutant TNFM2; provided by W. Lesslauer, F. Hoffman–La Roche²⁵ ). The double-point mutation in the TNFM1 mutant results in a minimal effect on the affinity of TNFM1 for TNFR1, whereas binding to TNFR2 is 1000-fold less. Similarly, the double-point mutation in the TNFM2 mutant results in a minimal effect on the affinity of TNFM2 for TNFR2, whereas binding to TNFR1 is 2500-fold less.²⁵ Isolated feline cardiac myocytes were used to assess the effects of the TNF- mutants for two reasons: first, the mechanical and electrophysiological properties of feline myocardium are similar to those of human subjects,²⁶ and second, it was not practical to obtain calcium-tolerant human cardiac myocytes from normal human hearts on a routine basis.

Cell motion was characterized with video edge detection as we have previously described in detail.³ Freshly isolated myocytes were treated for 30 minutes at 37°C with a single concentration of recombinant human TNF- (Genzyme) that is known to produce negative inotropic effects in isolated cardiac myocytes (0.1 nmol/L [200 U/mL³]) or a range of concentrations of TNFM1 (0.1 to 10 nmol/L), of TNFM2 (0.1 to 10 nmol/L), or a combination of TNFM1 (0.1 nmol/L) and TNFM2 (0.1 nmol/L, 10 nmol/L). Control cells were treated with an equal volume of diluent (10 mL/L [1%] of endotoxin-free human serum albumin). Results were expressed as the percentage change in cell length from resting values.²⁷

To confirm that the human TNF- mutants were capable of binding to TNF receptors in feline cardiac myocytes, we performed competition binding assays with the TNF- mutants using a modification of the method of Baglioni et al.²⁸ Briefly, 5x10³ freshly isolated cardiac myocytes were plated in 96-well plates in 200 µL of binding medium (Hanks' balanced salt solution supplemented with 50 mL/L [5%] fetal calf serum). The cells were incubated for 6 hours²⁸ at 4°C with 1 nmol/L of ¹²⁵I–TNF- in the presence of diluent, TNFM1 (60 nmol/L), or TNFM2 (60 nmol/L).²⁸ The myocytes were then removed from the plates and centrifuged at 20 000g for 2 minutes through phthalate mix (80% dibutyl phthalate oil, 20% olive oil) to separate bound from unbound ¹²⁵I–TNF-. The gamma emissions in the resultant cell pellets and supernatants were then determined. The amount of ¹²⁵I–TNF- binding to the cardiac myocytes was determined in the presence and absence of TNF- mutant ligands. Results for TNFM1 and TNFM2 were expressed as the percentage of total ¹²⁵I–TNF- binding, which was obtained in cells studied in the absence of a competing ligand. In control experiments (see "Appendix"), we confirmed the specificity of binding of the human TNF- mutants to feline TNFR1 and TNFR2.

Statistical Analysis
Each value is expressed as mean±SEM. One-way ANOVA was used to test for mean differences in cell shortening after treatment with TNF- or TNFM1 or TNFM2 mutants; where appropriate, post hoc multiple comparison testing was performed to test for differences between control and experimental groups (Dunnett's test) or between different experimental groups (Newman-Keuls). Significant differences were said to exist at P<.05.

	Results

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Myocardial TNF Receptors
TNF Receptor Gene Expression
Fig 1 shows a representative Northern blot of mRNA from three normal hearts with cDNA probes for human TNFR1 and TNFR2 and GAPDH. As shown, mRNA for both TNFR1 and TNFR2 was detected in each of the normal myocardial samples. Similar findings with respect to the gene expression for TNFR1 and TNFR2 were observed in the remaining two samples of normal human myocardium.

View larger version (66K): [in this window] [in a new window]   Figure 1. Tumor necrosis factor (TNF) receptor mRNA in normal myocardium. Total RNA was extracted from normal myocardium by the guanidinium thiocyanate method, and mRNA levels of TNF receptor proteins TNFR1 and TNFR2 were determined by Northern blot analysis with human probes for TNFR1 and TNFR2. GAPDH indicates human glyceraldehyde-3-phosphate dehydrogenase.

TNF Receptor Protein
Fig 2 shows the results of the TNFR1 and TNFR2 receptor protein assays in isolated membranes from whole heart homogenates of normal myocardium (n=5). As shown, both TNFR receptor subtypes were easily detected in solubilized human cardiac sarcolemmal preparations by receptor subtype–specific ELISA.

View larger version (11K): [in this window] [in a new window]   Figure 2. Bar graph of tumor necrosis factor (TNF) receptor proteins (TNFR1 and TNFR2) in normal myocardium. TNF receptor protein subtypes were measured in solubilized normal human cardiac sarcolemmal preparations with receptor subtype–specific ELISA (see "Methods" for details).

Immunolocalization of TNF Receptors
Fig 3A depicts the immunostaining pattern for normal myocardium with nonimmune rabbit sera (control) as the primary antibody, followed by a biotinylated goat anti-rabbit antibody. As shown, immunostaining with alkaline phosphatase was not observed when nonimmune serum was used. Fig 3B and 3C depicts the immunohistochemical staining patterns for normal human myocardium stained with anti-human TNFR1 and TNFR2 antibodies, respectively. The relevant finding is that the pattern of immunolocalization was similar for TNFR1 and TNFR2; that is, the immunostaining pattern for both receptors was localized to the cardiac myocytes. Histochemical analysis of other cell types in the myocardium revealed immunostaining of vascular smooth muscle cells, as well as the luminal border of vascular endothelial cells. In subsequent studies with monoclonal antibodies directed against TNFR1 and TNFR2, an identical staining pattern was obtained.

View larger version (0K): [in this window] [in a new window]   Figure 3. Photomicrographs of immunostaining of tumor necrosis factor (TNF) receptors TNFR1 and TNFR2 in normal myocardium. Immunostaining of human myocardial samples for TNFR1 and TNFR2 was performed with polyclonal anti-human antibodies against TNFR1 and TNFR2 (see "Methods" for details). A, Immunostaining of human myocardium when nonimmune serum was used as the source for the primary antibody. B and C, Immunohistochemical staining patterns for normal human myocardium using anti-human TNFR1 and TNFR2 antibodies, respectively.

Functional Assessment of TNF Receptors
The salient finding shown in Fig 4A is that the negative inotropic effects of wild-type TNF- were mimicked by the TNF- mutant that binds to TNFR1 (TNFM1), whereas the TNF- mutant that binds to TNFR2 (TNFM2) had no significant effect on cell motion, despite the use of a broad range of concentrations of the TNFM2 ligand (0.1 to 10 nmol/L). To test the possibility that TNFR1 might supply a cosignal that was necessary for TNFR2 to transduce negative inotropic effects, we stimulated the myocytes simultaneously with TNFM1 and TNFM2. Fig 4A (right) shows that simultaneous stimulation of the myocytes with TNFM1 (0.1 nmol/L) and TNFM2 (0.1 nmol/L, 10 nmol/L) did not produce a greater depression of cell shortening than was observed with stimulation with TNFM1 (0.1 nmol/L) alone. ANOVA indicated that there were overall significant differences in the extent of cell shortening between groups (P<.001); post hoc ANOVA testing (Dunnett's) indicated that there were significant differences from control (P<.05) values for wild-type TNF- and for each concentration of the TNFM1 tested, whereas values for TNFM2 were not significantly different (P>.05) from control. Furthermore, there was no significant difference (P>.05 [Newman-Keuls]) in the extent of cell shortening among groups when the results for stimulation with TNFM1 alone (0.1 nmol/L) were compared with results for simultaneous stimulation with TNFM1 (0.1 nmol/L) and TNFM2 (0.1 nmol/L, 10 nmol/L).

View larger version (40K): [in this window] [in a new window]   Figure 4. Bar graphs of functional significance and binding of tumor necrosis factor– (TNF-) mutants (TNFM1 and TNFM2) to feline cardiac myocytes. A, Cell motion studies with wild-type recombinant human TNF- and the human TNF- mutants. Freshly isolated cardiac myocytes were stimulated with wild-type recombinant human TNF-, and TNF mutants that bind selectively to human TNFR1 (corresponding mutant TNFM1) and to human TNFR2 (corresponding mutant TNFM2). Cell motion was characterized by video edge detection. Value for cell shortening in the control cells was 9.6±0.3%, which is similar to values we have reported previously.3 The concentrations for TNF-, TNFM1, and TNFM2 are depicted in nmol/L. *P<.05 compared with control values. B, Competitive binding assays for TNFM1 and TNFM2 in isolated feline cardiac myocytes (see "Methods" for details). Data for the binding assays shown in B represent the values (mean±SEM) from four different binding assays, each of which was performed in triplicate.

To confirm binding of the TNF- mutants to feline myocyte TNF receptors, competition binding assays were performed. Fig 4B shows that both TNF- mutants were able to compete with wild-type ¹²⁵I–TNF- for binding to isolated feline cardiac myocytes, as demonstrated by the decrease in ¹²⁵I–TNF- binding in the myocytes incubated with either TNFM1 (61.4±2.5% of control) or TNFM2 (46.7±4.5% of control).

	Discussion

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The present study shows that the adult human myocardium expresses both subtypes of TNF receptor: TNFR1 and TNFR2. Three separate lines of evidence support this statement. First, Northern blot analysis demonstrated TNFR1 and TNFR2 mRNA (Fig 1) in normal human myocardial samples. Second, both TNF receptor protein subtypes were easily detected in solubilized normal human cardiac sarcolemmal preparations by receptor subtype–specific ELISA (Fig 2). Third, immunohistochemical staining demonstrated the presence of TNFR1 and TNFR2 on the cardiac myocytes themselves.^{3 6 29} Although we are aware of one review that suggests that the myocardium binds radiolabeled TNF-,³⁰ we are unaware of previous reports that have delineated the specific presence of TNF receptors in adult cardiac myocytes. Thus, the demonstration of TNFR1 and TNFR2 on the cardiac myocytes provides a potential signaling pathway for the well-recognized deleterious negative inotropic effects of TNF-.^{9 29 31} However, although these studies were performed on myocardial samples from "normal" subjects with preserved left ventricular function (Table), we cannot exclude the possibility that the relative abundance of TNFR1 or TNFR2 mRNA and/or protein expression might have been influenced by changes in autonomic nervous system tone and/or other factors that might influence the activity of cytokines or the expression of their receptors.

A second major finding of the present study was that the negative inotropic effects of wild-type TNF- were mimicked by a TNF- mutant that binds to TNFR1, whereas stimulation of cardiac myocytes with the TNF- mutant that binds TNFR2 had no significant effect on cell motion. To test the possibility that TNFR1 might supply a cosignal that was necessary for TNFR2 to transduce negative inotropic effects, we simultaneously stimulated the myocytes with TNFM1 and TNFM2. However, simultaneous stimulation of the myocytes with TNFM1 and TNFM2 did not produce a greater depression of cell shortening than was observed with TNFM1 alone. Thus, taken together, these results suggest that the negative inotropic effects of TNF- are mediated by TNFR1, with little or no signaling contribution from TNFR2. Although we are unaware of a previous report that has characterized the effects of TNFR1 and TNFR2 on cardiac myocyte contractility, several recent indirect lines of evidence support the potential importance of TNFR1 in signaling the negative inotropic effects of TNF-. That is, previous studies in TNFR1-deficient transgenic mice have shown that these animals are resistant to the toxic effects of systemic TNF-³² as well as to the systemic toxicity of lipopolysaccharide or Staphylococcus aureus enterotoxin B.^{32 33} In contrast, comparable studies in TNFR2-deficient mice showed that although these animals were less sensitive to the toxic effects of systemic TNF-, they were not completely resistant to the deleterious effects of TNF- as were the TNFR1-deficient mice.³⁴ These latter findings have been interpreted as suggesting that TNFR2 "contributes" to TNF- toxicity by recruiting TNF- for interaction with TNFR1. This so-called "ligand passing"³⁵ between TNFR2 and TNFR1 might be expected to lower the concentration of TNF- needed for TNFR1 signal transduction.³⁴ Unfortunately, no mention was made in any of these reports^{32 33 34} with respect to the effects of TNF- or lipopolysaccharide on cardiac function. Finally, it should be recognized that our findings with respect to the effects of stimulating TNFR1 in cardiac myocytes are consistent with previous studies that show that the majority of the pleiotropic biological activities of TNF-, including the cytotoxic effects of this molecule, are signaled through TNFR1.^{36 37}

Despite the straightforward simplicity of the above findings with respect to the TNF- mutants, these results must be interpreted cautiously because the absolute specificity of binding of the human TNF- mutants to feline TNF receptors was not directly confirmed in the study. Finally, despite the similarities between feline and primate myocytes in terms of their calcium metabolism and mechanical and electrophysiological properties, it should be recognized that results obtained in feline myocytes may not be directly applicable to primate myocytes.

Conclusions
The elaboration of TNF- in destructive pathophysiological contexts, such as acute viral myocarditis,^{9 38} cardiac allograft rejection,^{10 39} myocardial infarction,^{40 41} myocardial reperfusion injury,⁴² and end-stage congestive heart failure,^{11 43} suggests that TNF- may play an important role in modulating the left ventricular dysfunction that supervenes in these conditions. Despite the potential importance of TNF- in these cardiac disease states, very little is known with respect to the basic mechanisms for the effects of TNF- in the heart. The results of the present study appear to be important for two reasons. First, the finding that cardiac myocytes express receptors for TNF- provides a rational framework for studying the signal transduction pathways that mediate the negative inotropic effects of TNF-, as well as for designing effective strategies to abrogate the negative inotropic effects of TNF-. Second, this study provides a basis for future studies, in which we will be able to determine whether TNF receptors are upregulated or downregulated in cardiac disease states in which TNF- is believed to play a deleterious role^{5 6 7 8 9 10 11 42} as well as to determine what might be the functional consequences of such TNF receptor regulation.

View larger version (16K): [in this window] [in a new window]   Figure 5. Plots of 125I–tumor necrosis factor– (TNF-) competition binding assays in human and feline peripheral blood mononuclear cells. A and B, Results for the saturation binding isotherms in human and feline peripheral blood mononuclear cells, respectively. Human and feline peripheral blood mononuclear leukocytes were harvested using Ficoll gradient centrifugation, and competition binding assays were performed for 6 hours28 at 4°C with 125I–TNF-, according to a modification of the method of Baglioni et al,28 as described in "Methods."

View larger version (14K): [in this window] [in a new window]   Figure 6. Bar graph of 125I–tumor necrosis factor– (TNF-) competition binding assays with TNFM1 and TNFM2 in human and feline peripheral blood mononuclear cells (PBMNs). Human and feline PBMNs were harvested using Ficoll gradient centrifugation, and competition binding assays were performed for 6 hours28 at 4°C with a single concentration of 125I–TNF- (1 nmol/L) in the presence of diluent (control), recombinant human (rh) TNF- (60 nmol/L), human TNFM1 (60 nmol/L), or human TNFM2 (60 nmol/L).28 Data are displayed as the percent of total binding of 125I–TNF- to human (open bar) and feline (closed bar) PBMNs in the presence and absence of specific competitors. As shown, recombinant human TNF-, TNFM1, and TNFM2 were able to bind competitively to human and feline TNF receptors to a similar degree. Data represent the mean±SEM of three separate binding assays from three separate primary myocyte isolations.

	Acknowledgments

 
This work was supported in part by funds from the Veterans Administration and by an educational grant from Merck and Company. The authors gratefully acknowledge the secretarial assistance of Jana Grana and Adrienne Chee and the technical assistance of Dorellyn Lee-Jackson, Faye Savafi, and Shaista Khan. We would like to thank Dr Andrew Schafer and Dr Robert Roberts for their ongoing support and guidance. Finally, we would like to thank both reviewers for their thoughtful criticisms of the manuscript.

	Footnotes

 
The Guest Editor was William H. Barry, MD, University of Utah Medical Center (Salt Lake City).

Two separate but interrelated control studies were performed to validate the use of the human TNF mutants in feline cells. In the initial series of experiments, we determined that the binding of human ¹²⁵I–TNF- was similar in human and feline peripheral mononuclear cells. For these studies, human and feline peripheral blood mononuclear leukocytes were harvested using Ficoll gradient centrifugation (Lymphocyte Separation Medium, Organon Teknika). Competition binding assays were performed with recombinant human ¹²⁵I–TNF- as described in "Methods."²⁸ Fig 5 shows two salient points with respect to these studies. First, there was saturable binding of recombinant human ¹²⁵I–TNF- to both human and feline peripheral blood mononuclear cells. Second, the binding affinities for ¹²⁵I–TNF were very similar for human (K_d=2.7x10^-10) and feline (K_d=3.4x10^-10) peripheral blood mononuclear cells. In the second series of experiments, we examined the competitive binding of TNF mutants in feline and human peripheral blood mononuclear cells. Fig 6, which is a summary of the results of these studies, demonstrates that the degree of competitive binding of TNFM2 to peripheral blood mononuclear cells was greater than for TNFM1, consistent with the fact that TNFR2 is the predominant cell type in peripheral blood mononuclear cells.²³ Moreover, this figure shows that the degrees of competitive binding for TNFM1 and TNFM2 were similar in the feline and human cells. Taken together, the results show that binding of recombinant human TNF- is similar in human and feline peripheral blood mononuclear cells and that the binding of the human TNF mutants is similar in human and feline peripheral blood mononuclear cells. Thus, these studies provide indirect data for the specificity of the binding of the human TNF- mutants to feline TNFR1 and TNFR2.

Received August 3, 1994; revision received March 15, 1995; accepted March 26, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Carswell EA, Old LJ, Kassel RL, Green S, Fiore N, Williamson B. An endotoxin-induced serum factor that causes necrosis of tumors. Proc Natl Acad Sci U S A. 1975;72:3666-3670. [Abstract/Free Full Text]
   
2. Gulick TS, Chung MK, Pieper SJ, Lange LG, Schreiner GF. Interleukin 1 and tumor necrosis factor inhibit cardiac myocyte ß-adrenergic responsiveness. Proc Natl Acad Sci U S A. 1989;86:6753-6757. [Abstract/Free Full Text]
   
3. Yokoyama T, Vaca L, Rossen RD, Durante W, Hazarika P, Mann DL. Cellular basis for the negative inotropic effects of tumor necrosis factor-alpha in the adult mammalian heart. J Clin Invest. 1993;92:2303-2312.
   
4. Schulz R, Nava E, Moncada S. Induction and potential biological relevance of a Ca^2+-independent nitric oxide synthase in the myocardium. Br J Pharmacol. 1992;105:575-580. [Medline] [Order article via Infotrieve]
   
5. Tracey KJ, Beutler B, Lowry SF, Merryweather J, Wolpe S, Milsark IW, Hariri RJ, Fahey TJ III, Zentella A, Albert JD, Shires GT. Shock and tissue injury induced by recombinant human cachectin. Science. 1986;234:470-474. [Abstract/Free Full Text]
   
6. Natanson C, Eichenholz PW, Danner RL, Eichacker W, Hoffman D, Kuo SM, Banks TJ, MacViottie TJ, Parrillo JE. Endotoxin and tumor necrosis factor challenges in dogs simulate the cardiovascular profile of human septic shock. J Exp Med. 1989;169:823-832. [Abstract/Free Full Text]
   
7. Parrillo JE, Parker MM, Natanson C, Suffredini AF, Danner RL, Cunnion RE, Ognibene FP. Septic shock in humans: advances in the understanding of pathogenesis, cardiovascular dysfunction, and therapy. Ann Intern Med. 1990;113:227-242.
   
8. Tracey KJ, Fong Y, Hesse DG, Manogue KR, Lee AT, Kuo GC, Lowry SF, Cerami A. Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature. 1987;330:662-664. [Medline] [Order article via Infotrieve]
   
9. Smith SC, Allen PM. Neutralization of endogenous tumor necrosis factor ameliorates the severity of myosin-induced myocarditis. Circ Res. 1992;70:856-863. [Abstract/Free Full Text]
   
10. Arbustini E, Grasso M, Diegoli M, Bramerio M, Foglieni AS, Albertario M, Matinelli L, Gavazzi A, Goggi C, Campana C, Vigano M. Expression of tumor necrosis factor in human acute cardiac rejection: an immunohistochemical and immunoblotting study. Am J Pathol. 1991;139:709-715. [Abstract]
    
11. Levine B, Kalman J, Mayer L, Fillit HM, Packer M. Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N Engl J Med. 1990;223:236-241.
    
12. Schall TJ, Lewis M, Koller KJ, Lee A, Rice GC, Wong GHW, Gatanaga T, Granger GA, Lentz R, Raab H, Kohr WJ, Goeddel DV. Molecular cloning and expression of a receptor for human tumor necrosis factor. Cell. 1990;61:361-370. [Medline] [Order article via Infotrieve]
    
13. Smith CA, Davis T, Anderson D, Solam L, Beckmann MP, Jerzy R, Dower SK, Cosman D, Goodwin RG. A receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins. Science. 1990;248:1019-1023. [Abstract/Free Full Text]
    
14. Gray PW, Barret K, Chantry D, Turner M, Feldmann M. Cloning of human necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein. Proc Natl Acad Sci U S A. 1990;87:7380-7384. [Abstract/Free Full Text]
    
15. Loetscher H, Pan YCE, Lahm HW, Gentz R, Brockhaus M, Tabuchi H, Lesslauer W. Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor. Cell. 1990;61:352-359.
    
16. Rothe J, Gehr G, Loetscher H, Lesslauer W. Tumor necrosis factor receptors: structure and function. Immunol Res. 1992;11:81-90. [Medline] [Order article via Infotrieve]
    
17. Vilcek J, Lee TH. Tumor necrosis factor: new insights into the molecular mechanisms of its multiple actions. J Biol Chem. 1991;266:7313-7316. [Free Full Text]
    
18. Ginsburg R, Bristow MR, Billingham ME, Stinson EB, Schroeder JS, Harrison DC. Study of the normal and failing isolated human heart: decreased response of failing heart to isoproterenol. Am Heart J. 1983;106:535-540. [Medline] [Order article via Infotrieve]
    
19. Bristow MR, Ginsburg R, Umans V, Fowler MB, Minobe W, Rasmussen R, Zera P, Menlove R, Shah P, Jamieson S, Stinson EB. ß₁- and ß₂-adrenergic-receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective ß₁-receptor downregulation in heart failure. Circ Res. 1986;59:297-309. [Abstract/Free Full Text]
    
20. Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: A Laboratory Approach. Cold Spring Harbor, NY: Cold Spring Harbor; 1989.
    
21. Stauber GB, Aiyer RA, Aggarwal BB. Human tumor necrosis factor- receptor: purification by immunoaffinity chromatography and initial characterization. J Biol Chem. 1988;263:19098-19104. [Abstract/Free Full Text]
    
22. Aggarwal BB, Graff K, Samal B, Higuchi M, Liao WSL. Regulation of two forms of the TNF receptors by phorbol ester and dibutyryl cyclic adenosine 3',5'-monophosphate in human histiocytic lymphoma cell line U-937. Lymphokine Cytokine Res. 1993;12:149-158. [Medline] [Order article via Infotrieve]
    
23. Winzen R, Wallach D, Engelmann H, Nophar Y, Brakebusch C, Kemper O, Resch K, Holtmann H. Selective decrease in cell surface expression and mRNA level of the 55-kDa tumor necrosis factor receptor during differentiation of HL-60 cells into macrophage-like but not granulocyte-like cells. J Immunol. 1992;148:3454-3460. [Abstract]
    
24. Yelavarthi K, Hunt JS. Analysis of p60 and p80 tumor necrosis factor- receptor messenger RNA and protein in human placentas. Am J Pathol. 1993;143:1131-1141. [Abstract]
    
25. Loetscher H, Stueber D, Banner D, Mackay F, Lesslauer W. Human tumor necrosis factor (TNF) mutants with exclusive specificity for the 55-kDa or 75-kDa TNF receptors. J Biol Chem. 1993;268:26350-26357. [Abstract/Free Full Text]
    
26. Cooper G, Tomanek RJ. Model dependent behaviour of pressure hypertrophied myocardium. Cardiovasc Res. 1987;21:342-351. [Medline] [Order article via Infotrieve]
    
27. Kapadia S, Torre-Amione G, Yokoyama T, Mann DL. Soluble tumor necrosis factor binding proteins modulate the negative inotropic effects of TNF- in vitro. Am J Physiol. 1995;37:H517-H525.
    
28. Baglioni C, McCandless S, Tavernier J, Fiers W. Binding of human necrosis factor to high affinity receptors on HeLa and lymphoblastoid cells sensitive to growth inhibition. J Biol Chem. 1985;260:13395-13397. [Abstract/Free Full Text]
    
29. Finkel MS, Oddis CV, Jacob TD, Watkins SC, Hattler BG, Simmons RL. Negative inotropic effects of cytokines on the heart mediated by nitric oxide. Science. 1992;257:387-389. [Abstract/Free Full Text]
    
30. Smith RS, Baglioni C. Characterization of TNF receptors. In: Aggarwal BB, Vilcek J, eds. Tumor Necrosis Factors. New York, NY: Marcel Dekker, Inc; 1990:131-147.
    
31. Giroir BP, Beutler B. Effect of amrinone on tumor necrosis factor production in endotoxin shock. Circ Shock. 1992;36:200-207. [Medline] [Order article via Infotrieve]
    
32. Rothe J, Lesslauer W, Lotscher H, Lang Y, Althage A, Zinkernagel R, Steinmetz M, Bluethmann H. Mice lacking the tumor necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature. 1993;364:798-802.[Medline] [Order article via Infotrieve]
    
33. Pfeffer K, Matsuyama T, Kundig TM, Wajeham A, Kishihara K, Shahinian A, Wiegmann K, Ohashi PS, Kronke M, Mak TW. Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxin shock, yet succumb to L. monocytogenes infection. Cell. 1993;73:457-467. [Medline] [Order article via Infotrieve]
    
34. Erickson S, de Sauvage FJ, Kikly K, Carver-Moore K, Pitts-Meek S, Gillett N, Sheehan CF, Schreiber RD, Goeddel DV, Moore MW. Decreased sensitivity to tumor-necrosis factor but normal T-cell development in TNF receptor-2-deficient mice. Nature. 1994;372:560-563. [Medline] [Order article via Infotrieve]
    
35. Tartaglia LA, Pennica D, 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. 1993;268:18542-18548. [Abstract/Free Full Text]
    
36. Engelmann H, Holtmann H, Brakebusch C, Avni YS, Sarov I, Nophar Y, Hadas E, Leitner O, Wallach D. Antibodies to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-like activity. J Biol Chem. 1990;265:14497-14504. [Abstract/Free Full Text]
    
37. Tartaglia LA, Weber RF, Figari IS, Reynolds C, Palladino MA, Goeddel DV. The two different receptors for tumor necrosis factor mediate distinct cellular responses. Proc Natl Acad Sci U S A. 1991;88:9292-9296. [Abstract/Free Full Text]
    
38. Matsumori A, Yamada T, Kawai C. Immunomodulating therapy in viral myocarditis: effects of tumour necrosis factor, interleukin 2 and anti-interleukin-2 receptor antibody in an animal model. Eur Heart J. 1991;12:203-205. [Abstract/Free Full Text]
    
39. Sakobar A, Horton JD, Bowler K, Manning R, Meager A, Dark JH. Raised levels of tumor-necrosis factor alpha (TNF) in cardiac transplant patients. Clin Transplant. 1993;7:459-466.
    
40. Maury CPJ, Teppo AM. Circulating tumour necrosis factor- (cachectin) in myocardial infarction. J Intern Med. 1989;225:333-336. [Medline] [Order article via Infotrieve]
    
41. Basaran Y, Basaran MM, Babacan KF, Ener B, Okay T, Gok H, Ozdemir M. Serum tumor necrosis factor levels in acute myocardial infarction and unstable angina pectoris. J Vasc Dis. 1993;332-337.
    
42. Lefer AM, Tsao P, Aoki N, Palladino MA Jr. Mediation of cardioprotection by transforming growth factor-ß. Science. 1990;249:61-64. [Abstract/Free Full Text]
    
43. Dutka DP, Elborn JS, Delamere F, Shale DJ, Morris GK. Tumour necrosis factor-alpha in severe congestive cardiac failure. Br Heart J. 1993;70:141-143.[Abstract/Free Full Text]
    

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