Purification of Type I and Type II Tumor Necrosis Factor Receptors from Human Lung Tissue Rasul Abdolrasulnia and Virginia L. Shepherd Department of Veterans Affairs Medical Center, and Departments of Medicine and Biochemistry, Vanderbilt University, Nashville, Tennessee
Two receptors for tumor necrosis factor-a (TNF) were purified from detergent-solubilized human lung tissues by adsorption to TNF-Sepharose, followed by elution with low pH. By SDS-PAGE analysis, the two proteins had molecular weights of75 and 55 leD. Using a soluble receptor assay, a binding affinity of approximately 1.2 nM was calculated for the isolated lung receptors. Each protein, isolated by electroelution from polyacrylamide gels, specifically bound TNF. Antibodies raised against the mixture of type I and II receptors bound specifically to both purified receptors by immunoblot analysis. Both the 75- and 55-leDreceptors could be precipitated from '25I-surface-Iabeled or 35S-methionine-labeled U937 cells using TNF-Sepharose or anti-receptor antibodies. In addition, the anti-TNF receptor antibodies partially blocked binding of TNF to U937 cells and specifically immunoprecipitated 125I-TNF cross-linked to its receptors on U937 cells. These results demonstrate that both type I and II TNF receptors can be isolated from human lung tissue by ligand affinity chromatography, and that U937 cells express both TNF receptor types.
'Iumor necrosis factor-a (TNF) is an important mediator of inflammation that is secreted predominantly by macrophages in response to bacterial lipopolysaccharide (LPS) (1). TNF has a variety of effects on both transformed and nontransformed cells that appear to be mediated by specific cell surface receptors (2-8). In most cells, a single class of receptors exists with an affinity constant of approximately 1 nM, and 1,000 to 10,000 sites per cell. Multiple receptor sizes were predicted by the many early cross-linking studies (2, 9-14). Recently, Hohmann and colleagues (15) reported the first evidence for the existence of more than one TNF receptor on HL-60 and U937 cells. Further studies from the same laboratory (16) and Brockhaus and associates (17) have clearly demonstrated the existence of at least two TNF receptors. Hohmann and colleagues (15) concluded that cells of myeloid origin express predominantly a type A (II) receptor with an approximate molecular weight of 75 leD, whereas cells of epithelial origin express a type B (I) receptor (55 kD). The cDNAs encoding both receptors
(Received in original form January 9, 1991 and in revised form December 20, 1991) Address correspondence to: Virginia L. Shepherd, Ph.D., VA Medical Center/Research, 1310 24th Ave. South, Nashville, TN 37212. Abbreviations: bovine serum albumin, BSA; disuccinimidyl suberate, DSS; horseradish peroxidase, HRP; immunoprecipitation buffer, IP buffer; lipopolysaccharide, LPS; phosphate-buffered saline, PBS; phenylmethylsulfonyl fluoride, PMSF; sodium dodecyl sulfate, SDS; sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE; 'Iris-buffered saline, TBS; tumor necrosis factor-a, TNF. Am. J. Respir. Cell Mol. BioI. Vol. 7. pp, 42-48, 1992
have been cloned and sequenced by several groups (18-23), and show < 25 % homology. The role ofTNF in regulating normal homeostatic conditions in the lung as well as its importance as a mediator of lung injury or inflammation is not known. No studies to date have demonstrated that TNF plays an important role in vivo in the lung, although several in vitro and in vivo studies have suggested that TNF might function as a key modulator of inflammation (24-28). In this study, we report the first isolation of both TNF receptors from human lung tissue using ligand affinity chromatography. Using antibodies prepared against these receptors, we have further demonstrated that the monocyte-like cell line U937 expresses both type I and II receptors.
Materials and Methods Materials Human recombinant TNF with a biologic activity of 2.4 X 107 U/mg was a gift from Cetus Corp. (Emeryville, CA). CNBr-activated Sepharose was purchased from Pharmacia (Piscataway, NJ). Human lung tissue was obtained from autopsy within 12 h post mortem. Carrier-free Na 125I was purchased from Amersham (Arlington Heights, IL). RPMI and DMEM media and fetal bovine serum were purchased from GIBCO (Grand Island, NY). Nuserum was from Collaborative Research (Lexington, MA). Lactoperoxidase, glucose oxidase, and Ficoll-Hypaque were purchased from Sigma Chemical Co. (St. Louis, MO). Disuccinimidyl suberate (DSS) was purchased from Pierce (Rockford, IL). Antisera raised against the soluble forms of the TNF receptors (TBP I and TBP II) were a gift from Dr. D. Wallach (Weizmann Institute) (29).
Abdolrasulinia and Shepherd: Human Lung TNF Receptors
Cell Cultures U937 cells were purchased from the American Type Culture Collection (Rockville, MD) and grown in suspension culture in RPMI medium with 10% Nuserum. Rat bone marrow macrophages were prepared as previously described (30). Iodination of TNF TNF (2.5 {.tg) was added to 100 {.tl of 0.1 M potassium phosphate buffer, pH 7.4, in a 12 X 75-mm plastic tube. Na 1251 (0.5 mCi in 5 {.tl) was added. Chloramine-T (10 {.tg in 30 {.tl) was added, and the mixture was incubated for 5 min on ice. The reaction was stopped by addition of 2-mercaptoethanol. The labeled TNF was separated from unbound iodine using a 5-ml Sephadex G-25 disposable column (Pharmacia) equilibrated in 10 mM Hepes buffer, pH 7.4, containing 0.15 M NaCl and 0.1% bovine serum albumin (BSA). The labeled TNF fractions were combined, with a resulting specific activity of approximately 2 X 108 cpm/{.tg of TNF. Th~ iodinated TNF retained biologic activity as measured usmg an L-cell cytotoxicity assay (31). Preparation of TNF-Sepharose TNF was coupled to CNBr-activated Sepharose according to the procedure supplied by the manufacturer. Briefly, 300 {.tg of TNF were coupled to CNBr-activated Sepharose (l g) by incubation for 2 h in 0.1 M NaHC03 buffer, pH 8.3, containing 0.5 M NaCl. Routinely, > 90% of the added TNF was covalently linked to the Sepharose. The TNF-Sepharose was washed, and any remaining reactive groups were blocked by incubation in 0.1 M Tris acetate buffer, pH 8.3, with 0.5 M NaCl. The immobilized TNF retained biologic activity as measured by the L-cell cytotoxicity assay (31) (data not shown). In addition, anti-TNF antibodies completely blocked the cytotoxicity, and BSA-Sepharose that had been prepared in an identical manner to TNF-Sepharose was not cytotoxic. Purification of the TNF Receptor The TNF receptor was isolated from human lung tissue obtained from autopsy using a modification of the procedure reported for purification of the human lung mannose receptor (32). All operations were carried out at 4 0 C unless otherwise specified. Briefly, human lung tissue (300 g) was homogenized in 20 mM Hepes buffer, pH 7.8, containing 10 mM MgC!" 2 mM EDTA, and 0.2 mM phenylmethylsulfonyl fluoride (PMSF) in a Waring blender. The homogenate was centrifuged at 16,500 X g for 30 min. The pellet was resuspended in solubilization buffer (20 mM Hepes, pH 7.8, containing 0.2 M NaCl, 20 mM CaCI2, 0.02 % sodium azide, and 0.2 mM PMSF) and rehomogenized. Triton X-100 was added to a final concentration of2 %, and the mixture was stirred for 4 h and then centrifuged at 16,500 X g for 30 min. The supernatant was brought to 20 mM CaCI2, added to 5 ml ofTNF-Sepharose in solubilization buffer, and incubated overnight at 4 0 C. The TNF receptor was eluted with 50 mM sodium acetate buffer, pH 3.0, containing 0.15 M NaCl. Fractions of 400 J.tl were collected directly into 200 {.tl of 0.25 M Hepes buffer, pH 8.2, containing 0.2 M NaCl, 20 mM CaC!" and 0.02 % sodium azide to immediately raise the pH of the TNF receptor fractions to pH 7.0 to 7.2. Fractions were tested for 12SI_TNF binding activity as de-
43
scribed below and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Purification of Type I and II TNF Receptors by SDS-PAGE and Electroelution Type I and II receptors were separated by SDS-PAGE followed by electroelution using a Centrilutor Micro-electroeluter (Amicon, Danvers, MA). Briefly, aliquots ofreceptor (25 {.tl) were electrophoresed under reducing conditions on a 7.5% SDS gel. The gel slices containing each receptor were cut from the gel and electroeluted separately into a Centricon microconcentrator at room temperature according to the Centrilutor operating instructions. The eluted proteins were concentrated by centrifugation in Centricon-30 microconcentrators at 4,000 X g for 10 min at 4 0 C. Analysis of the separated type I and II receptors by SDS-PAGE followed by silver staining showed that each receptor was > 95% pure (data not shown). Assay of 12SI_TNF Binding Activity of Purified Receptor The TNF-binding activity of the purified receptor was determined as follows: aliquots of fractions containing TNF receptor were incubated for 2 h at room temperature with 5 ng of 1251-TNF in 20 mM Tris, pH 7.2, containing 0.15 M NaCI and 0.1% BSA in a total volume of 500 {.tl. Ammonium sulfate (500 {.tl of an 80% solution) was added, and the solutions were incubated for 10 min on ice. The precipitated receptor-ligand complex was collected on glass fiber filters. The filters were washed with cold 40 % ammonium sulfate, dried, and counted in a gamma counter. A typical elution pattern is shown in Figure 1.
o z
80
z
~;;:) 60
!Il,
u.O zI- x
HE 40
:Q -
c.
u
v~
i:i:
8
20
Q.. (f)
o
4
6
8
FRACTION -#
Figure 1. TNF-Sepharose affinity purification of the TNF receptors from human lung tissue. Human lung tissue was homogenized and extracted with detergent as described in MATERIALS AND METHODS. The extract was incubated with TNF-Sepharose overnight at 4 0 C. TNF receptors were eluted with a pH 3.0 buffer, then immediately neutralized. The TNF binding activity was measured on an aliquot of each fraction as follows: the TNF receptor fraction was incubated for 2 h at room temperature with 12sI_TNF. TNFreceptor complexes were precipitated with 40% ammonium sulfate, and the precipitate collected on GF/C filters. The filters were washed, dried, and counted in a gamma counter. Companion assays were run with a 100-fold excess of unlabeled TNF to determine specific binding. Results are plotted as specific binding versus fraction number.
44
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL.
Determination of the Binding Affinity of the Purified Lung TNF Receptor Aliquots of receptor (approximately 1 ",g) were incubated with a constant amount of radioiodinated TNF (l X 106 cpm/5 ng/5 ",1) and increasing amounts of unlabeled TNF ranging from 0 to 10 ",g. The mixtures were incubated at room temperature for 2 h, then the receptor-ligand complex was precipitated with ammonium sulfate as described above. The binding affinity was calculated by the method of DeBlasi and co-workers (33). Binding of 12sl_TNF to Intact U937 Cells U937 cells (5 x 105) were incubated with 1251-TNF (5 ng) in a total volume of 0.1 ml in Hanks' balanced salt solution containing 1% BSA. Nonspecific binding was determined by the addition of a 100-fold excess of unlabeled TNF to companion tubes. The mixture was incubated for 2 h on ice, then cells and media were separated by centrifugation through oil. Cell-associated counts in the pellet were quantitated by gamma counting. Surface Labeling of U937 Cells with Na l2S1 and Isolation of TNF Receptors with TNF-Sepharose U937 cells (107) were suspended in 1 ml of 1 mM Hepes buffer, pH 7.4, containing 0.15 M NaCl. Glucose oxidase (3 U/25 ",1), lactoperoxidase (2 U/25 ",1), glucose (10 mM), and Na l251 (0.2 mCi) were added, and the mixture was incubated for 30 min on ice. The labeled cells were collected by centrifugation and washed 5 times with Hepes/NaCI. The cells were solubilized in phosphate-buffered saline (PBS) containing 0.5 % Triton X-lOO and incubated overnight with TNF-Sepharose (100 ",1 of packed gel). The TNF-Sepharose was pelleted and washed twice with PBSIO.5% Triton. The TNF-Sepharose was boiled in SDS-PAGE sample buffer for 10 min and electrophoresed on a 7.5 % reducing SDS gel. The electrophoresed proteins were fixed in 10 % acetic acidl7 % methanol. The gel was then dried, and the resulting radioactive receptor bands were visualized after exposure of Kodak X-OMAT film. The molecular weights of the protein bands were calculated using the relative mobility of 14C_ labeled protein standards. Preparation of Anti-receptor Antibodies Affinity-purified TNF receptor (approximately 20 ",g) was emulsified in Freund's complete adjuvant and injected subcutaneously into New Zealand white rabbits. The animals were boosted subcutaneously at 4 wk with 10 JJ-g of receptor in Freund's incomplete adjuvant. Immunoblot Analysis Purified receptor was electrophoresed under reducing conditions on 7.5% SDS gels. The proteins were electrophoretically transferred to nitrocellulose paper. The nitrocellulose was washed in Tris-buffered saline (TBS), then incubated for 1 h with TBS containing 0.5 % BSA. The blot was incubated overnight at 4 0 C with either a 1:100dilution of anti-receptor antiserum or preimmune serum. After extensive washing with TBS, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was added at a 1:2,000 dilution and the blot incubated for 2 h at room temperature. The paper was
71992
washed, and the reactive bands visualized by the HRPcatalyzed oxidation of 4-chloro-l-naphthol. Immunoprecipitation of the TNF Receptors from Surface-labeled U937 Cells 25 ! 1-Labeled cells were solubilized in 0.5 ml of immunoprecipitation buffer (IP buffer) (20 mM Tris, pH 7.75, containing 0.15 M NaCl, 1% Triton, 0.5 % deoxycholate, 0.02% sodium azide, and 0.34 U/ml of aprotinin). The solubilized membranes were precleared by incubation with preimmune serum (50 ",1),followed by addition of protein A-Sepharose (50 ",1 of a 10% suspension). Primary antiserum (50 ",1) was added to the supernatant, and the mixture incubated overnight at 4 0 C. Protein A-Sepharose was added, and the mixture incubated at room temperature for 30 min. The pellet was recovered by centrifugation and washed 3 times in wash buffer warmed to 37 0 C (10 mM Tris, pH 8.6, containing 0.15 M NaCl, 1 mM EDTA, 1% Triton, I% deoxycholate, 0.1% SDS, and 0.2 U aprotinin/ml). The immunoprecipitate was solubilized, boiled in SDS sample buffer, and analyzed by SDS-PAGE. The ! 251-labeled bands were visualized after overnight exposure of Kodak X-OMAT film. Covalent Cross-linking of TNF to the Cell Surface Receptor(s) on U937 Cells and Immunoprecipitation by Anti-TNF Receptor Antibodies DSS was freshly prepared as a 100-mM stock solution in dimethyl sulfoxide. 1251_TNF was covalently cross-linked to U937 cells as follows: cells were incubated with 25 ng oflabeled TNF for 2 h at 4 0 C in the presence or absence of a 100-fold excess of unlabeled TNF. Unbound TNF was removed by washing. The cells were then incubated with 1 mM DSS in 500 ",1 of PBS at 4 0 C for 60 min. The cells were washed and then solubilized in IP buffer for immunoprecipitation with anti-TNF receptor antibodies as described above. Counts associated with the immunoprecipitates were quantitated by gamma counting.
Results Purification of the Human TNF Receptor Detergent extracts of human lung tissue were incubated with TNF-Sepharose, and bound receptor was eluted with a low pH buffer. Fractions were analyzed for their ability to bind 25 ! 1-TNF (Figure 1). SDS-PAGE analysis of an aliquot of the combined active fractions is shown in Figure 2. Two bands of equal intensity at approximate molecular weights of 75 and 55 kD were routinely seen. Approximately 1 to 10 ",g of TNF receptor was purified from 300 g of tissue. Similar proteins have been isolated from human placental tissue, and, as with lung tissue, approximately equal amounts of both receptors were purified (data not shown). Measurement of the Binding Affinity of the Soluble TNF Receptor Using the soluble receptor assay described in MATERIALS AND METHODS, the binding affinity of the purified receptor was measured. Displacement of labeled TNF by increasing concentrations of unlabeled TNF is shown in Figure 3. From these data, we calculated a K, of approximately 1.2 nM using the method described by DeBlasi and co-workers (33).
Abdolrasulinia and Shepherd: Human Lung TNF Receptors
45
TABLE 1
Binding of 125I-TNF by the isolated 75- and 55-kD TNF receptors* 125I-TNF Bound" Receptor
--75kD
(cpm)
75 kD + 55 kD 75 kD 55 kD
* This is a
4,067 (1,712)* 2,175 (533) 7,616 (3,125)
representative experiment of two separate experiments.
t TNF binding is expressed as the total cpm bound by receptor plus TNF,
minus the cpm bound in the presence of a lOO-fold excess of unlabeled TNF. Each value is the average of duplicate determinations; numbers in parentheses indicate the range of the duplicates.
*
--55kD
Figure 2. SDS-PAGE analysis of the purified TNF receptors. Active fractions from the TNF-Sepharose column were combined and dialyzed extensively against 10 mM Hepes to bring the pH to 7.4. An aliquot (25 /-11) was then mixed with SDS sample buffer containing 2-mercaptoethanol and electrophoresed on 7.5% acrylamide gels. The protein bands were visualized after silver staining. The receptor bands are marked by the arrows and correspond to molecular weights of approximately 75 and 55 kD. Molecular weight standards are shown on the left.
Binding of TNF to the Receptors Isolated by Electroelution Each receptor was isolated from SDS gels by electroelution, and the binding of iodinated TNF was measured using the soluble binding assay. Each band specifically bound TNF as shown in Table 1.
Immunoblot Analysis Antibodies against the purified lung receptors were raised in rabbits. Using immunoblot analysis, these antibodies specifically recognized both type I and II receptors in the purified receptor preparation (Figure 4). To determine if these lung-derived receptors were identical to the previously reported receptors cloned from a variety of sources, we obtained antibodies from Dr. D. Wallach that were raised against the soluble TNF-binding proteins isolated from urine (29) and that reacted specifically against the type I and type II intact receptors. By immunoblot analysis, anti-TBP I reacted with the 55-kD lung receptor, and anti-TBP II reacted with the 75-kD receptor (data not shown). This supports the conclusion that the two TNF receptors isolated from human lung are immunologically similar to the previously described TNF receptors, and that the anti-lung TNF
0
z 80
......:75
:::> 0
!D
u, Z
I-;-
......
II'l
60
......:55
40
~
u,
0
*
20 0
2 LOG [TNF]
A
B
Figure 3. Competition by increasing concentrations of unlabeled
Figure 4. Immunoblot analysis of the purified type I and type II
TNF for binding of 125I_TNF to the purified receptor. Receptor (25 /-11) was incubated with 5 ng oflabeled TNF plus increasing concentrations of unlabeled TNF in the soluble assay described in Figure 1. Receptor-TNF complexes were collected on filters and counted. The results are expressed as the percentage of 125I_TNF displaced with increasing concentrations of added TNF. The data are averages of duplicate determinations and are representative ofthree separate experiments.
TNF receptors from human lung. TNF receptor (25 /-11) was electrophoresed under reducing conditions on a 7.5% SDS polyacrylamide gel. The proteins were electrophoretically transferred to nitrocellulose. The nitrocellulose paper was incubated in preimmune serum (lane A) or primary antibody (lane B) overnight, and reactive bands were visualized as described in MATERIALS AND METHODS. Molecular weight standards are marked by the small arrowheads.
46
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 71992
receptor antibodies specifically react with the same type I and II receptors. Ligand Precipitation and Immunoprecipitation of the TNF Receptors from U937 Cells Intact U937 cells were surface-labeled with Na 125I, then solubilized and incubated with TNF-Sepharose or antireceptor antiserum as described in MATERIALS AND METHODS. The precipitated proteins were analyzed by SDS-PAGE. Both TNF-Sepharose and antibody specifically precipitated both receptors (75 and 55 kD) from U937 cells (Figure 5). Immunoprecipitation of 125I-Labeled TNF Covalently Cross-linked to Its Receptor(s) on U937 Cells l25I-TNF was chemically cross-linked to U937 cells as described in MATERIALS AND METHODS. The cells plus TNF were solubilized and incubated with anti-lung receptor antibodies or preimmune serum. After extensive washing, the amount of immunoprecipitated TNF-receptor complex was quantitated by gamma counting. As shown in Table 2, antilung TNF receptor antiserum specifically precipitated the covalently linked TNF receptor complex from U937 cells. Similar data has also been obtained from rat bone marrow-derived macrophages and human monocyte-derived macrophages (data not shown).
TABLE 2
Immunoprecipitation of 1251_TNF covalently cross-linked to its receptor on U937 cells Immunoprecipitable TNF Receptor (cpm)
Experiment 1 l25I-TNF 125I-TNF + excess unlabeled TNF Experiment 2 125I-TNF 125I-TNF + excess TNF
1,308 162 2,642 235
Inhibition of Binding of 125I_TNF to U937 Cells by Anti-lung Receptor Antibodies U937 cells express both the type I and type II TNF receptor as described above. To further substantiate the identity of the purified lung receptors as the type I and type II TNF receptors, the ability of the anti-lung receptor antiserum to inhibit ligand binding to U937 cells was examined. As shown in Figure 6, preimmune serum had no effect on TNF binding, whereas the anti-lung receptor antibody inhibited binding by approximately 40%.
Discussion In this study, we report for the first time the purification of both type I and type II TNF receptors from human lung tissue by affinity chromatography on TNF-Sepharose. By a number of criteria, we have demonstrated that these proteins are identical to the previously cloned type I and type II TNF receptors. Both receptors displayed similar molecular weights reported by other groups for the cell surface TNF receptors (75 and 55 kD). The isolated receptors bound TNF
75kD~
55kD~
3000
E
Q,
CJ
0
z
1
~
2
Figure 5. Isolation of the TNF receptor from U937 cells using TNF-Sepharose precipitation and immunoprecipitation. Lane 1: Solubilized,surface-labeledU937 cells were precleared by incubation with preimmuneserum for 60 min at room temperature. Protein A-Sepharose wasthen addedfor 30 min, and the Sepharose was removedby centrifugation. Rabbit anti-human TNF receptor (100 JLl) was added, and the mixture incubated overnight at 4 C. The antibody-antigen complex was collected by precipitation with protein A-Sepharose, and analyzed on 7.5% SDS gels under reducing conditions. The radioactive bands were visualized after exposure of Kodak X-OMAT film. Lane 2: U937 cells were surface-labeled with Na125I, solubilized in detergent, and incubated with TNFSepharose overnight at 4 0 C. The receptor-TNF-Sepharose complex was boiled in SDS sample buffer, then electrophoresed on a 7.5% acrylamide gel. The radioactive receptor bands were visualized as above. The arrowheads mark the positionsof the molecular weight standards used in Figure 2. 0
2000
0 m LL
z
I-
1000
o C
Ab Pre
Figure 6. U937 cells (5 x 105) were incubated with 125I-labeled TNF (5 ng) for 2 h at 4 C in buffer alone (C), or in the presence of added anti-TNF receptor antiserum (1:100 dilution) (Ab) or preimmune serum (1:100 dilution) (Pre). Companiontubes for each condition containing a 100-fold excess of unlabeled TNF were run to determine nonspecific binding. Results are the average specific binding ± the standard deviation of triplicate determinations. These data are representative of two separate experiments. 0
Abdolrasulinia and Shepherd: Human Lung TNF Receptors
in a soluble receptor assay with an affinity of 1.2 nM, similar to the K, found for TNF binding to U937 cells. The purified receptors were capable of blocking TNF-mediated killing of L cells. Antibodies raised against these proteins reacted with the purified lung receptors by immunoblot analysis. Both type I and type II receptors were specifically precipitated from surface-labeled U937 cells using immobilized ligand and anti-receptor antibodies. Finally, the antihuman lung TNF receptor antibody partially blocked TNF binding to U937 cells and specifically immunoprecipitated labeled TNF cross-linked to the receptor on U937 cells. From previous studies by Hohmann and colleagues (15) and Brockhaus and associates (17), the reports of the molecular cloning of two distinct TNF receptor cDNAs (18-23), and the purification of two receptors reported in the present study, it is now well established that at least two types of TNF receptors exist, and that these receptors are products of two distinct genes. We have shown that normal lung tissue (a mixture of many cell types) and the macrophage-like cell line U937 express both receptors. In addition, receptor purified from placental tissue likewise is a mixture of both receptors (data not shown). This is supported by the findings of Smith and co-workers (22) and Gray and colleagues (20) that cDNA probes for both receptors react with placental mRNA by Northern analyses. Results from other studies (15) suggest that monocytes and mature macrophages express predominantly the 75-kD receptor. However, in this study, U937 cells of the monocyte lineage were found to express both receptors in approximately equal quantities, and other laboratories have reported that human monocytes, HL60 cells, and U937 cells express mRNA for both receptors (19, 22, 34). The function of two distinct TNF receptors is unknown. Although it has been shown that some cells express predominantly one type of TNF receptor, such as monocytes, it has been reported that these same cells express the mRNA for the other receptor. Therefore, it is possible that cells have the capacity to express both receptor proteins, and that regulation at the level of translation might determine which type of receptor is expressed. This is supported by the studies of Hohmann and colleagues (35), who reported that dibutyryl cAMP increased expression of the type II receptor, with no change in the type I receptor. It has been reported that TNF signaling involves a variety of transduction mechanisms (36-38). Each receptor type might be linked to a distinct signaling system, and differential regulation of these receptors might lead to modulation and control of the target cell response. To our knowledge, this is the first report of the isolation of a cytokine receptor from human lung tissue. The fact that TNF receptors can be isolated from normal lung tissue suggests that TNF might playa role in the maintenance of normal lung homeostasis. In addition, recent evidence from our laboratory showing regulation of receptor levels on lung-specific cells by agents such as LPS, interferon-y or interleukin-l (V. Shepherd, unpublished results) would imply that the response to TNF mediated by target cell receptors can be modulated by other cytokines and inflammatory mediators. Acknowledgments: the writers thank Dr. Charles Harlan, Davidson County Medical Examiner, for tissue from autopsies. This work was supported by the Department of Veterans Affairs.
47
References I. Larrick, J. W., and S. L. Kunkel. 1988. The role of tumor necrosis factor and interleukin I in the irnmunoinflammatory response. Pharm. Res.
5:129-139. 2. Kull, F. c., Jr., S. Jacobs, and P. Cuatrecasas. 1985. Cellular receptors for 125I_labeled tumor necrosis factor: specific binding, affinitylabeling, and relationship to sensitivity. Proc. Natl. Acad. Sci. USA 82:57565760. 3. Tsujimoto, M., Y. K. Yip, and J. Vilcek. 1985. Tumor necrosis factor: specific binding and internalization in sensitive and resistant cells. Proc. Natl. Acad. Sci. USA 82:7626-7630. 4. Baglioni, C., S. McCandless, J. Tavernier, and W. Fiers. 1985. Binding of human tumor necrosis factor to high affinity receptors on HeLa and 1ymphoblastoid cells sensitive to growth inhibition. J. Bioi. Chem. 260:13395-13397. 5. Imamura, K., D. Spriggs, and D. Kufe. 1987. Expression of tumor necrosis factor receptors on human monocytes and internalization of receptor bound ligand. J. Immunol. 139:2989-2992. 6. Nawroth, P. P., 1. Bank, D. Handley, J. Cassismeris, L. Chess, and D. Stern. 1986. Tumor necrosis factor/cachectin interacts with endothelial cell receptors to induce release of interleukin 1. J. Exp. Med. 163: 1363-1375. 7. Vilcek, J., M. Tsujimoto, V. J. Palombella, M. Kohase, and J. Le. 1987. Tumor necrosis factor: receptor binding and mitogenic action in fibroblasts. J. Cell. Physiol. (Suppl.) 5:57-61. 8. Scheurich, P., B. Thoma, U. Ucer, and K. Pfizenmaier. 1987. Immunoregulatory activity of recombinant human tumor necrosis factor (TNF)alpha: induction of TNF receptors on human T cells and TNFalpha-mediated enhancement of T cell responses. J. Immunol. 138: 1786-1792. 9. Scheurich, P., U. Ucer, M. Kronke, and K. Pfizenmaier. 1986. Quantification and characterization of high-affinity membrane receptor for tumor necrosis factor on human leukemic cell lines. Int. J. Cancer 38:127-133. 10. Smith, R. A., M. Kirstein, W. Piers, and C. Baglioni. 1986. Species specificity of human and murine tumor necrosis factor. J. Bioi. Chem. 261:14871-14874. 11. Tsujimoto, M., R. Feinman, M. Kohase, and 1. Vilcek. 1986. Characterization and affinity crosslinking of receptors for tumor necrosis factor on human cells. Arch. Biochem. Biophys. 249:563-568. 12. Yoshie, 0., K. Tada, and N. Ishida. 1986. Binding and crosslinking of l2SI-labe1ed recombinant human tumor necrosis factor to cell surface receptors. J. Biochem. 100:531-541. 13. Creasey, A. A., R. Yamamoto, and C. R. Vitt. 1987. A high molecular weight component of the human tumor necrosis factor receptor is associated with cytotoxicity. Proc. Natl. Acad. Sci. USA 84:3293-3297. 14. Stauber, G. B., R. A. Aiyer, and B. B. Aggarwal. 1988. Human tumor necrosis factor-a receptor. J. Bioi. Chem. 263:19098-19104. 15. Hohmann, H.-P., R. Rerny, M. Brockhaus, and A. P. G. M. van Loon. 1989. Two different cell types have different major receptors for human tumor necrosis factor (TNFa). J. BioI. Chem. 264:14927-14934. 16. Hohmann, H.-P., R. Remy, B. Posch!, and A. P. G. M. van Loon. 1990. Tumor necrosis factors-a and -{3 bind to the same two types of tumor necrosis factor receptors and maximally activate the transcription factor NF-KB at low receptor occupancy and within minutes after receptor binding. J. Bioi. Chem. 265:15183-15188. 17. Brockhaus, M., H.-J. Schoenfeld, E.-J. Sch!aeger, W. Hunziker, W. Lesslauer, and H. Loetscher. 1990. Identificationof two types of tumor necrosis factor receptors on human cell lines by monoclonal antibodies. Immunology 87:3127-3131. 18. Schall, T. J., M. Lewis, K. J. Koller et al. 1990. Molecular cloning and expression of a receptor for human tumor necrosis factor. Cell 61: 361-370. 19. Loetscher, H., Y.-C. E. Pan, H.-W. Lahm et al. 1990. Molecular cloning and expression of the human 55 kD tumor necrosis factor receptor. Cell 61:351-359. 20. Gray, P. W., K. Barrett, D. Chantry, M. Turner, and M. Feldmann. 1990. Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein. Proc. Natl. Acad. Sci. USA 87:7380-7384. 21. Nophar, Y., O. Kemper, C. Brakebusch et al. 1990. Soluble forms of tumor necrosis factor receptors (TNF-Rs). The cDNA for the type I TNF-R, cloned using amino acid sequence data of its soluble form, encodes both the cell surface and a soluble form of the receptor. EMBO J. 9:32693278. 22. Smith, C. A., T. Davis, D. Anderson et al. 1990. A receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins. Science 248: 1019-1023. 23. Heller, R. A., K. Song, M. A. Onasch, W. H. Fischer, D. Chang, and G. M. Ringold. 1990. Complementary DNA cloning ofa receptor for tumor necrosis factor and demonstration of a shed form of the receptor. Proc. Natl. Acad. Sci. USA 87:6151-6155. 24. Millar, A. B., M. Singer, A. Meager, N. M. Foley, N. M. Johnson, and G. A. W. Rook. 1989. Tumor necrosis factor in bronchopulmonary secre-
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25. 26. 27. 28. 29. 30.
31. 32.
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 7 1992
tions of patients with adult respiratory distress syndrome. Lancet 2: 712-714. Michie, H. R., K. R. Manogue, D. R. Spriggs et al. 1988. Detection of circulating tumor necrosis factor after endotoxin administration. N. Engl. J. Med. 318:1481-1486. Beutler, B., I. W. Milsarek, and A. C. Cerami. 1985. Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effects of endotoxin. Science 229:869-871. Tracey, K. J., J. Y. Fong, D. G. Hesseetal. 1987. Anti-TNFamonoclonal antibodies prevent septic shock during lethal bacteremia. Nature 330: 662-664. Fong, Y.,K. J. Tracey, L. L. Moldaweretal. 1989. Antibodies to cachectin/tumor necrosis factor reduce interleukin-I beta and interleukin-6 appearance during lethal bacteremia. J. Exp. Med. 170:1627-1633. Engelmann, H., D. Novick, and D. Wallach. 1990. Two tumor necrosis factor-binding proteins purified from human urine. J. Bioi. Chem. 265:1531-1536. Shepherd, V. L., M. G. Konish, and P. Stahl. 1985. Dexamethasone increases expression of mannose receptors and decreases extracellular lysosomal enzyme accumulation in macrophages. J. Bioi. Chem. 260: 160-164. Kirstein, M., W. Fiers , and C. Baglioni. 1986. Growth inhibition and cytotoxicity of tumor necrosis factor in L929 cells is enhanced by high cell density and inhibition of mRNA synthesis. J. Immunol. 137:2277-2280. Stephenson, J. D., and V. L. Shepherd. 1987. Purification of the human
33. 34.
35.
36.
37. 38.
alveolar macrophage mannose receptor. Biochem. Biophys. Res. Commun. 148:883-889. DeBlasi, A., K. O'Reilly, and H. J. Motulski. 1989. Calculating receptor number from binding experiments using same compound as radioligand and competitor. Trends Pharmacol. Sci. 10:227-229. Lindvall, L., M. Lantz, U. Gullberg, and I. Olsson. 1990. Modulation of the constitutive gene expression of the 55 kD tumor necrosis factor receptor in hematopoietic cells. Biochem. Biophys. Res. Commun. 172: 557-563. Hohmann, H. P., M. Brockhaus, P. A. Baeuerle, R. Remy, R. Kolbeck, and A. P. van Loon. 1990. Expression of the types A and B tumor necrosis factor (TNF) receptors is independently regulated, and both receptors mediate activation of the transcription factor NF-KB. J. Bioi. Chern. 265:22409-22415. Zhang, Y., J.-X. Lin, Y.K. Yip, andJ. Vilcek. 1988. Enhancement of cAMP levels and protein kinase activity by tumor necrosis factor and interleukin I in human fibroblasts: role in the induction of interleukin 6. Proc. Natl. Acad. Sci. USA 85:6802-6806. Palombella, F. C., and J. Vilcek. 1989. Mitogenic and cytotoxic actions of tumor necrosis factor in BALB/c 3T3 cells. Role of phospholipase activation. J. Bioi. Chem. 264: 18128-18133. Hayakawa, M., T. Hori, S. Shibamoto, M. Tsjimoto, N. Oku, and F. Ito. 1991. Solubilization of human placental tumor necrosis factor receptors as a complex with a guanine nucleotide-binding protein. Arch. Biochem. Biophys. 286:323-329.
Two receptors for tumor necrosis factor-a (TNF) were purified from detergent-solubilized human lung tissues by adsorption to TNF-Sepharose, followed by elution with low pH. By SDS-PAGE analysis, the two proteins had molecular weights of75 and 55 leD. Using a soluble receptor assay, a binding affinity of approximately 1.2 nM was calculated for the isolated lung receptors. Each protein, isolated by electroelution from polyacrylamide gels, specifically bound TNF. Antibodies raised against the mixture of type I and II receptors bound specifically to both purified receptors by immunoblot analysis. Both the 75- and 55-leDreceptors could be precipitated from '25I-surface-Iabeled or 35S-methionine-labeled U937 cells using TNF-Sepharose or anti-receptor antibodies. In addition, the anti-TNF receptor antibodies partially blocked binding of TNF to U937 cells and specifically immunoprecipitated 125I-TNF cross-linked to its receptors on U937 cells. These results demonstrate that both type I and II TNF receptors can be isolated from human lung tissue by ligand affinity chromatography, and that U937 cells express both TNF receptor types.
'Iumor necrosis factor-a (TNF) is an important mediator of inflammation that is secreted predominantly by macrophages in response to bacterial lipopolysaccharide (LPS) (1). TNF has a variety of effects on both transformed and nontransformed cells that appear to be mediated by specific cell surface receptors (2-8). In most cells, a single class of receptors exists with an affinity constant of approximately 1 nM, and 1,000 to 10,000 sites per cell. Multiple receptor sizes were predicted by the many early cross-linking studies (2, 9-14). Recently, Hohmann and colleagues (15) reported the first evidence for the existence of more than one TNF receptor on HL-60 and U937 cells. Further studies from the same laboratory (16) and Brockhaus and associates (17) have clearly demonstrated the existence of at least two TNF receptors. Hohmann and colleagues (15) concluded that cells of myeloid origin express predominantly a type A (II) receptor with an approximate molecular weight of 75 leD, whereas cells of epithelial origin express a type B (I) receptor (55 kD). The cDNAs encoding both receptors
(Received in original form January 9, 1991 and in revised form December 20, 1991) Address correspondence to: Virginia L. Shepherd, Ph.D., VA Medical Center/Research, 1310 24th Ave. South, Nashville, TN 37212. Abbreviations: bovine serum albumin, BSA; disuccinimidyl suberate, DSS; horseradish peroxidase, HRP; immunoprecipitation buffer, IP buffer; lipopolysaccharide, LPS; phosphate-buffered saline, PBS; phenylmethylsulfonyl fluoride, PMSF; sodium dodecyl sulfate, SDS; sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE; 'Iris-buffered saline, TBS; tumor necrosis factor-a, TNF. Am. J. Respir. Cell Mol. BioI. Vol. 7. pp, 42-48, 1992
have been cloned and sequenced by several groups (18-23), and show < 25 % homology. The role ofTNF in regulating normal homeostatic conditions in the lung as well as its importance as a mediator of lung injury or inflammation is not known. No studies to date have demonstrated that TNF plays an important role in vivo in the lung, although several in vitro and in vivo studies have suggested that TNF might function as a key modulator of inflammation (24-28). In this study, we report the first isolation of both TNF receptors from human lung tissue using ligand affinity chromatography. Using antibodies prepared against these receptors, we have further demonstrated that the monocyte-like cell line U937 expresses both type I and II receptors.
Materials and Methods Materials Human recombinant TNF with a biologic activity of 2.4 X 107 U/mg was a gift from Cetus Corp. (Emeryville, CA). CNBr-activated Sepharose was purchased from Pharmacia (Piscataway, NJ). Human lung tissue was obtained from autopsy within 12 h post mortem. Carrier-free Na 125I was purchased from Amersham (Arlington Heights, IL). RPMI and DMEM media and fetal bovine serum were purchased from GIBCO (Grand Island, NY). Nuserum was from Collaborative Research (Lexington, MA). Lactoperoxidase, glucose oxidase, and Ficoll-Hypaque were purchased from Sigma Chemical Co. (St. Louis, MO). Disuccinimidyl suberate (DSS) was purchased from Pierce (Rockford, IL). Antisera raised against the soluble forms of the TNF receptors (TBP I and TBP II) were a gift from Dr. D. Wallach (Weizmann Institute) (29).
Abdolrasulinia and Shepherd: Human Lung TNF Receptors
Cell Cultures U937 cells were purchased from the American Type Culture Collection (Rockville, MD) and grown in suspension culture in RPMI medium with 10% Nuserum. Rat bone marrow macrophages were prepared as previously described (30). Iodination of TNF TNF (2.5 {.tg) was added to 100 {.tl of 0.1 M potassium phosphate buffer, pH 7.4, in a 12 X 75-mm plastic tube. Na 1251 (0.5 mCi in 5 {.tl) was added. Chloramine-T (10 {.tg in 30 {.tl) was added, and the mixture was incubated for 5 min on ice. The reaction was stopped by addition of 2-mercaptoethanol. The labeled TNF was separated from unbound iodine using a 5-ml Sephadex G-25 disposable column (Pharmacia) equilibrated in 10 mM Hepes buffer, pH 7.4, containing 0.15 M NaCl and 0.1% bovine serum albumin (BSA). The labeled TNF fractions were combined, with a resulting specific activity of approximately 2 X 108 cpm/{.tg of TNF. Th~ iodinated TNF retained biologic activity as measured usmg an L-cell cytotoxicity assay (31). Preparation of TNF-Sepharose TNF was coupled to CNBr-activated Sepharose according to the procedure supplied by the manufacturer. Briefly, 300 {.tg of TNF were coupled to CNBr-activated Sepharose (l g) by incubation for 2 h in 0.1 M NaHC03 buffer, pH 8.3, containing 0.5 M NaCl. Routinely, > 90% of the added TNF was covalently linked to the Sepharose. The TNF-Sepharose was washed, and any remaining reactive groups were blocked by incubation in 0.1 M Tris acetate buffer, pH 8.3, with 0.5 M NaCl. The immobilized TNF retained biologic activity as measured by the L-cell cytotoxicity assay (31) (data not shown). In addition, anti-TNF antibodies completely blocked the cytotoxicity, and BSA-Sepharose that had been prepared in an identical manner to TNF-Sepharose was not cytotoxic. Purification of the TNF Receptor The TNF receptor was isolated from human lung tissue obtained from autopsy using a modification of the procedure reported for purification of the human lung mannose receptor (32). All operations were carried out at 4 0 C unless otherwise specified. Briefly, human lung tissue (300 g) was homogenized in 20 mM Hepes buffer, pH 7.8, containing 10 mM MgC!" 2 mM EDTA, and 0.2 mM phenylmethylsulfonyl fluoride (PMSF) in a Waring blender. The homogenate was centrifuged at 16,500 X g for 30 min. The pellet was resuspended in solubilization buffer (20 mM Hepes, pH 7.8, containing 0.2 M NaCl, 20 mM CaCI2, 0.02 % sodium azide, and 0.2 mM PMSF) and rehomogenized. Triton X-100 was added to a final concentration of2 %, and the mixture was stirred for 4 h and then centrifuged at 16,500 X g for 30 min. The supernatant was brought to 20 mM CaCI2, added to 5 ml ofTNF-Sepharose in solubilization buffer, and incubated overnight at 4 0 C. The TNF receptor was eluted with 50 mM sodium acetate buffer, pH 3.0, containing 0.15 M NaCl. Fractions of 400 J.tl were collected directly into 200 {.tl of 0.25 M Hepes buffer, pH 8.2, containing 0.2 M NaCl, 20 mM CaC!" and 0.02 % sodium azide to immediately raise the pH of the TNF receptor fractions to pH 7.0 to 7.2. Fractions were tested for 12SI_TNF binding activity as de-
43
scribed below and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Purification of Type I and II TNF Receptors by SDS-PAGE and Electroelution Type I and II receptors were separated by SDS-PAGE followed by electroelution using a Centrilutor Micro-electroeluter (Amicon, Danvers, MA). Briefly, aliquots ofreceptor (25 {.tl) were electrophoresed under reducing conditions on a 7.5% SDS gel. The gel slices containing each receptor were cut from the gel and electroeluted separately into a Centricon microconcentrator at room temperature according to the Centrilutor operating instructions. The eluted proteins were concentrated by centrifugation in Centricon-30 microconcentrators at 4,000 X g for 10 min at 4 0 C. Analysis of the separated type I and II receptors by SDS-PAGE followed by silver staining showed that each receptor was > 95% pure (data not shown). Assay of 12SI_TNF Binding Activity of Purified Receptor The TNF-binding activity of the purified receptor was determined as follows: aliquots of fractions containing TNF receptor were incubated for 2 h at room temperature with 5 ng of 1251-TNF in 20 mM Tris, pH 7.2, containing 0.15 M NaCI and 0.1% BSA in a total volume of 500 {.tl. Ammonium sulfate (500 {.tl of an 80% solution) was added, and the solutions were incubated for 10 min on ice. The precipitated receptor-ligand complex was collected on glass fiber filters. The filters were washed with cold 40 % ammonium sulfate, dried, and counted in a gamma counter. A typical elution pattern is shown in Figure 1.
o z
80
z
~;;:) 60
!Il,
u.O zI- x
HE 40
:Q -
c.
u
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i:i:
8
20
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o
4
6
8
FRACTION -#
Figure 1. TNF-Sepharose affinity purification of the TNF receptors from human lung tissue. Human lung tissue was homogenized and extracted with detergent as described in MATERIALS AND METHODS. The extract was incubated with TNF-Sepharose overnight at 4 0 C. TNF receptors were eluted with a pH 3.0 buffer, then immediately neutralized. The TNF binding activity was measured on an aliquot of each fraction as follows: the TNF receptor fraction was incubated for 2 h at room temperature with 12sI_TNF. TNFreceptor complexes were precipitated with 40% ammonium sulfate, and the precipitate collected on GF/C filters. The filters were washed, dried, and counted in a gamma counter. Companion assays were run with a 100-fold excess of unlabeled TNF to determine specific binding. Results are plotted as specific binding versus fraction number.
44
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL.
Determination of the Binding Affinity of the Purified Lung TNF Receptor Aliquots of receptor (approximately 1 ",g) were incubated with a constant amount of radioiodinated TNF (l X 106 cpm/5 ng/5 ",1) and increasing amounts of unlabeled TNF ranging from 0 to 10 ",g. The mixtures were incubated at room temperature for 2 h, then the receptor-ligand complex was precipitated with ammonium sulfate as described above. The binding affinity was calculated by the method of DeBlasi and co-workers (33). Binding of 12sl_TNF to Intact U937 Cells U937 cells (5 x 105) were incubated with 1251-TNF (5 ng) in a total volume of 0.1 ml in Hanks' balanced salt solution containing 1% BSA. Nonspecific binding was determined by the addition of a 100-fold excess of unlabeled TNF to companion tubes. The mixture was incubated for 2 h on ice, then cells and media were separated by centrifugation through oil. Cell-associated counts in the pellet were quantitated by gamma counting. Surface Labeling of U937 Cells with Na l2S1 and Isolation of TNF Receptors with TNF-Sepharose U937 cells (107) were suspended in 1 ml of 1 mM Hepes buffer, pH 7.4, containing 0.15 M NaCl. Glucose oxidase (3 U/25 ",1), lactoperoxidase (2 U/25 ",1), glucose (10 mM), and Na l251 (0.2 mCi) were added, and the mixture was incubated for 30 min on ice. The labeled cells were collected by centrifugation and washed 5 times with Hepes/NaCI. The cells were solubilized in phosphate-buffered saline (PBS) containing 0.5 % Triton X-lOO and incubated overnight with TNF-Sepharose (100 ",1 of packed gel). The TNF-Sepharose was pelleted and washed twice with PBSIO.5% Triton. The TNF-Sepharose was boiled in SDS-PAGE sample buffer for 10 min and electrophoresed on a 7.5 % reducing SDS gel. The electrophoresed proteins were fixed in 10 % acetic acidl7 % methanol. The gel was then dried, and the resulting radioactive receptor bands were visualized after exposure of Kodak X-OMAT film. The molecular weights of the protein bands were calculated using the relative mobility of 14C_ labeled protein standards. Preparation of Anti-receptor Antibodies Affinity-purified TNF receptor (approximately 20 ",g) was emulsified in Freund's complete adjuvant and injected subcutaneously into New Zealand white rabbits. The animals were boosted subcutaneously at 4 wk with 10 JJ-g of receptor in Freund's incomplete adjuvant. Immunoblot Analysis Purified receptor was electrophoresed under reducing conditions on 7.5% SDS gels. The proteins were electrophoretically transferred to nitrocellulose paper. The nitrocellulose was washed in Tris-buffered saline (TBS), then incubated for 1 h with TBS containing 0.5 % BSA. The blot was incubated overnight at 4 0 C with either a 1:100dilution of anti-receptor antiserum or preimmune serum. After extensive washing with TBS, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was added at a 1:2,000 dilution and the blot incubated for 2 h at room temperature. The paper was
71992
washed, and the reactive bands visualized by the HRPcatalyzed oxidation of 4-chloro-l-naphthol. Immunoprecipitation of the TNF Receptors from Surface-labeled U937 Cells 25 ! 1-Labeled cells were solubilized in 0.5 ml of immunoprecipitation buffer (IP buffer) (20 mM Tris, pH 7.75, containing 0.15 M NaCl, 1% Triton, 0.5 % deoxycholate, 0.02% sodium azide, and 0.34 U/ml of aprotinin). The solubilized membranes were precleared by incubation with preimmune serum (50 ",1),followed by addition of protein A-Sepharose (50 ",1 of a 10% suspension). Primary antiserum (50 ",1) was added to the supernatant, and the mixture incubated overnight at 4 0 C. Protein A-Sepharose was added, and the mixture incubated at room temperature for 30 min. The pellet was recovered by centrifugation and washed 3 times in wash buffer warmed to 37 0 C (10 mM Tris, pH 8.6, containing 0.15 M NaCl, 1 mM EDTA, 1% Triton, I% deoxycholate, 0.1% SDS, and 0.2 U aprotinin/ml). The immunoprecipitate was solubilized, boiled in SDS sample buffer, and analyzed by SDS-PAGE. The ! 251-labeled bands were visualized after overnight exposure of Kodak X-OMAT film. Covalent Cross-linking of TNF to the Cell Surface Receptor(s) on U937 Cells and Immunoprecipitation by Anti-TNF Receptor Antibodies DSS was freshly prepared as a 100-mM stock solution in dimethyl sulfoxide. 1251_TNF was covalently cross-linked to U937 cells as follows: cells were incubated with 25 ng oflabeled TNF for 2 h at 4 0 C in the presence or absence of a 100-fold excess of unlabeled TNF. Unbound TNF was removed by washing. The cells were then incubated with 1 mM DSS in 500 ",1 of PBS at 4 0 C for 60 min. The cells were washed and then solubilized in IP buffer for immunoprecipitation with anti-TNF receptor antibodies as described above. Counts associated with the immunoprecipitates were quantitated by gamma counting.
Results Purification of the Human TNF Receptor Detergent extracts of human lung tissue were incubated with TNF-Sepharose, and bound receptor was eluted with a low pH buffer. Fractions were analyzed for their ability to bind 25 ! 1-TNF (Figure 1). SDS-PAGE analysis of an aliquot of the combined active fractions is shown in Figure 2. Two bands of equal intensity at approximate molecular weights of 75 and 55 kD were routinely seen. Approximately 1 to 10 ",g of TNF receptor was purified from 300 g of tissue. Similar proteins have been isolated from human placental tissue, and, as with lung tissue, approximately equal amounts of both receptors were purified (data not shown). Measurement of the Binding Affinity of the Soluble TNF Receptor Using the soluble receptor assay described in MATERIALS AND METHODS, the binding affinity of the purified receptor was measured. Displacement of labeled TNF by increasing concentrations of unlabeled TNF is shown in Figure 3. From these data, we calculated a K, of approximately 1.2 nM using the method described by DeBlasi and co-workers (33).
Abdolrasulinia and Shepherd: Human Lung TNF Receptors
45
TABLE 1
Binding of 125I-TNF by the isolated 75- and 55-kD TNF receptors* 125I-TNF Bound" Receptor
--75kD
(cpm)
75 kD + 55 kD 75 kD 55 kD
* This is a
4,067 (1,712)* 2,175 (533) 7,616 (3,125)
representative experiment of two separate experiments.
t TNF binding is expressed as the total cpm bound by receptor plus TNF,
minus the cpm bound in the presence of a lOO-fold excess of unlabeled TNF. Each value is the average of duplicate determinations; numbers in parentheses indicate the range of the duplicates.
*
--55kD
Figure 2. SDS-PAGE analysis of the purified TNF receptors. Active fractions from the TNF-Sepharose column were combined and dialyzed extensively against 10 mM Hepes to bring the pH to 7.4. An aliquot (25 /-11) was then mixed with SDS sample buffer containing 2-mercaptoethanol and electrophoresed on 7.5% acrylamide gels. The protein bands were visualized after silver staining. The receptor bands are marked by the arrows and correspond to molecular weights of approximately 75 and 55 kD. Molecular weight standards are shown on the left.
Binding of TNF to the Receptors Isolated by Electroelution Each receptor was isolated from SDS gels by electroelution, and the binding of iodinated TNF was measured using the soluble binding assay. Each band specifically bound TNF as shown in Table 1.
Immunoblot Analysis Antibodies against the purified lung receptors were raised in rabbits. Using immunoblot analysis, these antibodies specifically recognized both type I and II receptors in the purified receptor preparation (Figure 4). To determine if these lung-derived receptors were identical to the previously reported receptors cloned from a variety of sources, we obtained antibodies from Dr. D. Wallach that were raised against the soluble TNF-binding proteins isolated from urine (29) and that reacted specifically against the type I and type II intact receptors. By immunoblot analysis, anti-TBP I reacted with the 55-kD lung receptor, and anti-TBP II reacted with the 75-kD receptor (data not shown). This supports the conclusion that the two TNF receptors isolated from human lung are immunologically similar to the previously described TNF receptors, and that the anti-lung TNF
0
z 80
......:75
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u, Z
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60
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40
~
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0
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20 0
2 LOG [TNF]
A
B
Figure 3. Competition by increasing concentrations of unlabeled
Figure 4. Immunoblot analysis of the purified type I and type II
TNF for binding of 125I_TNF to the purified receptor. Receptor (25 /-11) was incubated with 5 ng oflabeled TNF plus increasing concentrations of unlabeled TNF in the soluble assay described in Figure 1. Receptor-TNF complexes were collected on filters and counted. The results are expressed as the percentage of 125I_TNF displaced with increasing concentrations of added TNF. The data are averages of duplicate determinations and are representative ofthree separate experiments.
TNF receptors from human lung. TNF receptor (25 /-11) was electrophoresed under reducing conditions on a 7.5% SDS polyacrylamide gel. The proteins were electrophoretically transferred to nitrocellulose. The nitrocellulose paper was incubated in preimmune serum (lane A) or primary antibody (lane B) overnight, and reactive bands were visualized as described in MATERIALS AND METHODS. Molecular weight standards are marked by the small arrowheads.
46
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 71992
receptor antibodies specifically react with the same type I and II receptors. Ligand Precipitation and Immunoprecipitation of the TNF Receptors from U937 Cells Intact U937 cells were surface-labeled with Na 125I, then solubilized and incubated with TNF-Sepharose or antireceptor antiserum as described in MATERIALS AND METHODS. The precipitated proteins were analyzed by SDS-PAGE. Both TNF-Sepharose and antibody specifically precipitated both receptors (75 and 55 kD) from U937 cells (Figure 5). Immunoprecipitation of 125I-Labeled TNF Covalently Cross-linked to Its Receptor(s) on U937 Cells l25I-TNF was chemically cross-linked to U937 cells as described in MATERIALS AND METHODS. The cells plus TNF were solubilized and incubated with anti-lung receptor antibodies or preimmune serum. After extensive washing, the amount of immunoprecipitated TNF-receptor complex was quantitated by gamma counting. As shown in Table 2, antilung TNF receptor antiserum specifically precipitated the covalently linked TNF receptor complex from U937 cells. Similar data has also been obtained from rat bone marrow-derived macrophages and human monocyte-derived macrophages (data not shown).
TABLE 2
Immunoprecipitation of 1251_TNF covalently cross-linked to its receptor on U937 cells Immunoprecipitable TNF Receptor (cpm)
Experiment 1 l25I-TNF 125I-TNF + excess unlabeled TNF Experiment 2 125I-TNF 125I-TNF + excess TNF
1,308 162 2,642 235
Inhibition of Binding of 125I_TNF to U937 Cells by Anti-lung Receptor Antibodies U937 cells express both the type I and type II TNF receptor as described above. To further substantiate the identity of the purified lung receptors as the type I and type II TNF receptors, the ability of the anti-lung receptor antiserum to inhibit ligand binding to U937 cells was examined. As shown in Figure 6, preimmune serum had no effect on TNF binding, whereas the anti-lung receptor antibody inhibited binding by approximately 40%.
Discussion In this study, we report for the first time the purification of both type I and type II TNF receptors from human lung tissue by affinity chromatography on TNF-Sepharose. By a number of criteria, we have demonstrated that these proteins are identical to the previously cloned type I and type II TNF receptors. Both receptors displayed similar molecular weights reported by other groups for the cell surface TNF receptors (75 and 55 kD). The isolated receptors bound TNF
75kD~
55kD~
3000
E
Q,
CJ
0
z
1
~
2
Figure 5. Isolation of the TNF receptor from U937 cells using TNF-Sepharose precipitation and immunoprecipitation. Lane 1: Solubilized,surface-labeledU937 cells were precleared by incubation with preimmuneserum for 60 min at room temperature. Protein A-Sepharose wasthen addedfor 30 min, and the Sepharose was removedby centrifugation. Rabbit anti-human TNF receptor (100 JLl) was added, and the mixture incubated overnight at 4 C. The antibody-antigen complex was collected by precipitation with protein A-Sepharose, and analyzed on 7.5% SDS gels under reducing conditions. The radioactive bands were visualized after exposure of Kodak X-OMAT film. Lane 2: U937 cells were surface-labeled with Na125I, solubilized in detergent, and incubated with TNFSepharose overnight at 4 0 C. The receptor-TNF-Sepharose complex was boiled in SDS sample buffer, then electrophoresed on a 7.5% acrylamide gel. The radioactive receptor bands were visualized as above. The arrowheads mark the positionsof the molecular weight standards used in Figure 2. 0
2000
0 m LL
z
I-
1000
o C
Ab Pre
Figure 6. U937 cells (5 x 105) were incubated with 125I-labeled TNF (5 ng) for 2 h at 4 C in buffer alone (C), or in the presence of added anti-TNF receptor antiserum (1:100 dilution) (Ab) or preimmune serum (1:100 dilution) (Pre). Companiontubes for each condition containing a 100-fold excess of unlabeled TNF were run to determine nonspecific binding. Results are the average specific binding ± the standard deviation of triplicate determinations. These data are representative of two separate experiments. 0
Abdolrasulinia and Shepherd: Human Lung TNF Receptors
in a soluble receptor assay with an affinity of 1.2 nM, similar to the K, found for TNF binding to U937 cells. The purified receptors were capable of blocking TNF-mediated killing of L cells. Antibodies raised against these proteins reacted with the purified lung receptors by immunoblot analysis. Both type I and type II receptors were specifically precipitated from surface-labeled U937 cells using immobilized ligand and anti-receptor antibodies. Finally, the antihuman lung TNF receptor antibody partially blocked TNF binding to U937 cells and specifically immunoprecipitated labeled TNF cross-linked to the receptor on U937 cells. From previous studies by Hohmann and colleagues (15) and Brockhaus and associates (17), the reports of the molecular cloning of two distinct TNF receptor cDNAs (18-23), and the purification of two receptors reported in the present study, it is now well established that at least two types of TNF receptors exist, and that these receptors are products of two distinct genes. We have shown that normal lung tissue (a mixture of many cell types) and the macrophage-like cell line U937 express both receptors. In addition, receptor purified from placental tissue likewise is a mixture of both receptors (data not shown). This is supported by the findings of Smith and co-workers (22) and Gray and colleagues (20) that cDNA probes for both receptors react with placental mRNA by Northern analyses. Results from other studies (15) suggest that monocytes and mature macrophages express predominantly the 75-kD receptor. However, in this study, U937 cells of the monocyte lineage were found to express both receptors in approximately equal quantities, and other laboratories have reported that human monocytes, HL60 cells, and U937 cells express mRNA for both receptors (19, 22, 34). The function of two distinct TNF receptors is unknown. Although it has been shown that some cells express predominantly one type of TNF receptor, such as monocytes, it has been reported that these same cells express the mRNA for the other receptor. Therefore, it is possible that cells have the capacity to express both receptor proteins, and that regulation at the level of translation might determine which type of receptor is expressed. This is supported by the studies of Hohmann and colleagues (35), who reported that dibutyryl cAMP increased expression of the type II receptor, with no change in the type I receptor. It has been reported that TNF signaling involves a variety of transduction mechanisms (36-38). Each receptor type might be linked to a distinct signaling system, and differential regulation of these receptors might lead to modulation and control of the target cell response. To our knowledge, this is the first report of the isolation of a cytokine receptor from human lung tissue. The fact that TNF receptors can be isolated from normal lung tissue suggests that TNF might playa role in the maintenance of normal lung homeostasis. In addition, recent evidence from our laboratory showing regulation of receptor levels on lung-specific cells by agents such as LPS, interferon-y or interleukin-l (V. Shepherd, unpublished results) would imply that the response to TNF mediated by target cell receptors can be modulated by other cytokines and inflammatory mediators. Acknowledgments: the writers thank Dr. Charles Harlan, Davidson County Medical Examiner, for tissue from autopsies. This work was supported by the Department of Veterans Affairs.
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References I. Larrick, J. W., and S. L. Kunkel. 1988. The role of tumor necrosis factor and interleukin I in the irnmunoinflammatory response. Pharm. Res.
5:129-139. 2. Kull, F. c., Jr., S. Jacobs, and P. Cuatrecasas. 1985. Cellular receptors for 125I_labeled tumor necrosis factor: specific binding, affinitylabeling, and relationship to sensitivity. Proc. Natl. Acad. Sci. USA 82:57565760. 3. Tsujimoto, M., Y. K. Yip, and J. Vilcek. 1985. Tumor necrosis factor: specific binding and internalization in sensitive and resistant cells. Proc. Natl. Acad. Sci. USA 82:7626-7630. 4. Baglioni, C., S. McCandless, J. Tavernier, and W. Fiers. 1985. Binding of human tumor necrosis factor to high affinity receptors on HeLa and 1ymphoblastoid cells sensitive to growth inhibition. J. Bioi. Chem. 260:13395-13397. 5. Imamura, K., D. Spriggs, and D. Kufe. 1987. Expression of tumor necrosis factor receptors on human monocytes and internalization of receptor bound ligand. J. Immunol. 139:2989-2992. 6. Nawroth, P. P., 1. Bank, D. Handley, J. Cassismeris, L. Chess, and D. Stern. 1986. Tumor necrosis factor/cachectin interacts with endothelial cell receptors to induce release of interleukin 1. J. Exp. Med. 163: 1363-1375. 7. Vilcek, J., M. Tsujimoto, V. J. Palombella, M. Kohase, and J. Le. 1987. Tumor necrosis factor: receptor binding and mitogenic action in fibroblasts. J. Cell. Physiol. (Suppl.) 5:57-61. 8. Scheurich, P., B. Thoma, U. Ucer, and K. Pfizenmaier. 1987. Immunoregulatory activity of recombinant human tumor necrosis factor (TNF)alpha: induction of TNF receptors on human T cells and TNFalpha-mediated enhancement of T cell responses. J. Immunol. 138: 1786-1792. 9. Scheurich, P., U. Ucer, M. Kronke, and K. Pfizenmaier. 1986. Quantification and characterization of high-affinity membrane receptor for tumor necrosis factor on human leukemic cell lines. Int. J. Cancer 38:127-133. 10. Smith, R. A., M. Kirstein, W. Piers, and C. Baglioni. 1986. Species specificity of human and murine tumor necrosis factor. J. Bioi. Chem. 261:14871-14874. 11. Tsujimoto, M., R. Feinman, M. Kohase, and 1. Vilcek. 1986. Characterization and affinity crosslinking of receptors for tumor necrosis factor on human cells. Arch. Biochem. Biophys. 249:563-568. 12. Yoshie, 0., K. Tada, and N. Ishida. 1986. Binding and crosslinking of l2SI-labe1ed recombinant human tumor necrosis factor to cell surface receptors. J. Biochem. 100:531-541. 13. Creasey, A. A., R. Yamamoto, and C. R. Vitt. 1987. A high molecular weight component of the human tumor necrosis factor receptor is associated with cytotoxicity. Proc. Natl. Acad. Sci. USA 84:3293-3297. 14. Stauber, G. B., R. A. Aiyer, and B. B. Aggarwal. 1988. Human tumor necrosis factor-a receptor. J. Bioi. Chem. 263:19098-19104. 15. Hohmann, H.-P., R. Rerny, M. Brockhaus, and A. P. G. M. van Loon. 1989. Two different cell types have different major receptors for human tumor necrosis factor (TNFa). J. BioI. Chem. 264:14927-14934. 16. Hohmann, H.-P., R. Remy, B. Posch!, and A. P. G. M. van Loon. 1990. Tumor necrosis factors-a and -{3 bind to the same two types of tumor necrosis factor receptors and maximally activate the transcription factor NF-KB at low receptor occupancy and within minutes after receptor binding. J. Bioi. Chem. 265:15183-15188. 17. Brockhaus, M., H.-J. Schoenfeld, E.-J. Sch!aeger, W. Hunziker, W. Lesslauer, and H. Loetscher. 1990. Identificationof two types of tumor necrosis factor receptors on human cell lines by monoclonal antibodies. Immunology 87:3127-3131. 18. Schall, T. J., M. Lewis, K. J. Koller et al. 1990. Molecular cloning and expression of a receptor for human tumor necrosis factor. Cell 61: 361-370. 19. Loetscher, H., Y.-C. E. Pan, H.-W. Lahm et al. 1990. Molecular cloning and expression of the human 55 kD tumor necrosis factor receptor. Cell 61:351-359. 20. Gray, P. W., K. Barrett, D. Chantry, M. Turner, and M. Feldmann. 1990. Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein. Proc. Natl. Acad. Sci. USA 87:7380-7384. 21. Nophar, Y., O. Kemper, C. Brakebusch et al. 1990. Soluble forms of tumor necrosis factor receptors (TNF-Rs). The cDNA for the type I TNF-R, cloned using amino acid sequence data of its soluble form, encodes both the cell surface and a soluble form of the receptor. EMBO J. 9:32693278. 22. Smith, C. A., T. Davis, D. Anderson et al. 1990. A receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins. Science 248: 1019-1023. 23. Heller, R. A., K. Song, M. A. Onasch, W. H. Fischer, D. Chang, and G. M. Ringold. 1990. Complementary DNA cloning ofa receptor for tumor necrosis factor and demonstration of a shed form of the receptor. Proc. Natl. Acad. Sci. USA 87:6151-6155. 24. Millar, A. B., M. Singer, A. Meager, N. M. Foley, N. M. Johnson, and G. A. W. Rook. 1989. Tumor necrosis factor in bronchopulmonary secre-
48
25. 26. 27. 28. 29. 30.
31. 32.
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 7 1992
tions of patients with adult respiratory distress syndrome. Lancet 2: 712-714. Michie, H. R., K. R. Manogue, D. R. Spriggs et al. 1988. Detection of circulating tumor necrosis factor after endotoxin administration. N. Engl. J. Med. 318:1481-1486. Beutler, B., I. W. Milsarek, and A. C. Cerami. 1985. Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effects of endotoxin. Science 229:869-871. Tracey, K. J., J. Y. Fong, D. G. Hesseetal. 1987. Anti-TNFamonoclonal antibodies prevent septic shock during lethal bacteremia. Nature 330: 662-664. Fong, Y.,K. J. Tracey, L. L. Moldaweretal. 1989. Antibodies to cachectin/tumor necrosis factor reduce interleukin-I beta and interleukin-6 appearance during lethal bacteremia. J. Exp. Med. 170:1627-1633. Engelmann, H., D. Novick, and D. Wallach. 1990. Two tumor necrosis factor-binding proteins purified from human urine. J. Bioi. Chem. 265:1531-1536. Shepherd, V. L., M. G. Konish, and P. Stahl. 1985. Dexamethasone increases expression of mannose receptors and decreases extracellular lysosomal enzyme accumulation in macrophages. J. Bioi. Chem. 260: 160-164. Kirstein, M., W. Fiers , and C. Baglioni. 1986. Growth inhibition and cytotoxicity of tumor necrosis factor in L929 cells is enhanced by high cell density and inhibition of mRNA synthesis. J. Immunol. 137:2277-2280. Stephenson, J. D., and V. L. Shepherd. 1987. Purification of the human
33. 34.
35.
36.
37. 38.
alveolar macrophage mannose receptor. Biochem. Biophys. Res. Commun. 148:883-889. DeBlasi, A., K. O'Reilly, and H. J. Motulski. 1989. Calculating receptor number from binding experiments using same compound as radioligand and competitor. Trends Pharmacol. Sci. 10:227-229. Lindvall, L., M. Lantz, U. Gullberg, and I. Olsson. 1990. Modulation of the constitutive gene expression of the 55 kD tumor necrosis factor receptor in hematopoietic cells. Biochem. Biophys. Res. Commun. 172: 557-563. Hohmann, H. P., M. Brockhaus, P. A. Baeuerle, R. Remy, R. Kolbeck, and A. P. van Loon. 1990. Expression of the types A and B tumor necrosis factor (TNF) receptors is independently regulated, and both receptors mediate activation of the transcription factor NF-KB. J. Bioi. Chern. 265:22409-22415. Zhang, Y., J.-X. Lin, Y.K. Yip, andJ. Vilcek. 1988. Enhancement of cAMP levels and protein kinase activity by tumor necrosis factor and interleukin I in human fibroblasts: role in the induction of interleukin 6. Proc. Natl. Acad. Sci. USA 85:6802-6806. Palombella, F. C., and J. Vilcek. 1989. Mitogenic and cytotoxic actions of tumor necrosis factor in BALB/c 3T3 cells. Role of phospholipase activation. J. Bioi. Chem. 264: 18128-18133. Hayakawa, M., T. Hori, S. Shibamoto, M. Tsjimoto, N. Oku, and F. Ito. 1991. Solubilization of human placental tumor necrosis factor receptors as a complex with a guanine nucleotide-binding protein. Arch. Biochem. Biophys. 286:323-329.
