(cid:9)
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`Thalidomide Selectively Inhibits Tumor
`Necrosis Factor ac Production by Stimulated
`Human Monocytes
`By Elizabeth P. Sampaio,* Euzenir N. Sarno, $ Ruth Galilly,*
`Zanvil A. Cohn,* and Gilla Kaplan*
`
`From the 'Laboratory of Cellular Physiology and Immunology, The Rockefeller University,
`New York, New York 10021; and the #Leprosy Unit, Osuatldo Cruz Foundation, Manguinhos,
`Rio deJaneiro, Brazil, 21045
`
`Summary
`Thalidomide selectively inhibits the production of human monocyte tumor necrosis factor a
`(TNF-a) when these cells are triggered with lipopolysaccharide and other agonists in culture.
`40% inhibition occurs at the clinically achievable dose of the drug of 1 Ag/ml. In contrast, the
`amount of total protein and individual proteins labeled with [31S]methionine and expressed on
`SDS-PAGE are not influenced. The amounts of interleukin 10 (11,1(3), Ilr6, and granulocyte/
`macrophage colony-stimulating factor produced by monorytes remain unaltered. The selectivity
`of this drug may be useful in determining the role of TNF-a in vivo and modulating its toxic
`effects in a clinical setting.
`
`Thalidomide (a-N-pthalimidoglutarimide) has a long phar-
`
`macological history, having been used as a sedative, an
`antiinflammatory, and an immunosuppressive agent (1-3).
`Currently, it is used for the therapy of erythema nodosum
`leprosum (ENL),' or type II reaction, an acute inflamma-
`tory state occurring in lepromatous leprosy characterized by
`severe systemic symptoms, including fever, painful cutaneous
`lesions, arthritis, glomerulonephritis, and the presence of cir-
`culating immune complexes (4). In this serious complication
`of leprosy, thalidomide has a prompt and dramatic effect,
`decreasing inflammation and enhancing patient well-being
`so that it remains the drug of choice for the therapy of ENL
`(5). The fever, weight loss, and general debility of ENL, as
`in other forms of acute and chronic disease, including sepsis
`and cancer, may be associated with the production of macro-
`phage-derived cytokines (6, 7). Recently, serum levels of
`TNF-a and 11,1/3 released mainly by mononuclear phago-
`cytes were found to be markedly elevated in ENL (8). In ad-
`dition, it was noted that TNF-a levels were reduced after
`treatment of patients in ENL. This prompted a detailed ex-
`amination of the effects of thalidomide on cytokine produc-
`tion by monorytes.
`
`'Abbreviations used in this paper. CWRML, cell wall protein of Mpco-
`bacterium kp ae ; ENL, erythema nodosum leprosum; GM-CSF, granulocyte/
`macrophage colony-stimulating factor; PPD, purified protein derivative
`of tuberculin.
`
`Materials and Methods
`PBMC obtained by Ficoll-Hypaque (Phar-
`Monocyte Isolation.
`macia Fine Chemicals, Piscataway, NJ) density centrifugation were
`rosetted with neuraminidase-treated (Vibrio cholerae neuraminidase;
`Calbiochem-Behring Corp., LaJolla, CA) sheep erythrocytes (Scott
`Laboratories, Friskville, RI) (SRBC rosetting), and the nonrosetted
`cells were counted (E - population monorytes enriched). 106 cells
`were cultured at 37°C in 24-well plates (Coming Glass Works,
`Coming, NY) in 1 ml of RPMI 1640 (Gibco Laboratories, Grand
`Island, NY) supplemented with 10% AB* serum, 100 U/ml pen-
`icillin, 100 Etg/ml streptomycin, and 2 MM L-glutamine. Adherent
`E - cells were used for the studies.
`LPS ofSalmonella minnesota R595 (List Bio-
`Cytokine Agonists.
`logical Laboratories, Campbell, CA) was diluted in PBS, pH 7.4,
`and used at 1 wg/ml; Purified protein derivative of tuberculin (PPD)
`was purchased from Statens Seruminstitut, Copenhagen, Denmark;
`cell wall protein of Mycobacterium leprae (CWP-ML) was prepared
`through the National Institute ofAllergy and Infectious Diseases,
`and provided by Dr. Patrick Brennan (Department ofMicrobiology,
`Colorado State University, Fort Collins, CO). Concentration of
`the stimulating agents were determined in previous experiments
`to induce optimal TNF-tx protein production by cultured mono-
`cytes. The endotoxin content of solutions and mycobacterial prepa-
`rations was estimated by the Limulus amebocyte lysate assay (LAL;
`Whittaker M.A. Bioproducts, Walkersville, MD). All solutions
`used contained <10 pg/ml of endotoxin.
`Adherent E- cells were stimulated with
`Cytokine Induction .
`1 hg/ml of LPS, 10 Rg/ml of PPD, or 10 Wg/ml of CWP-ML for
`up to 18-20 h. At various times, supernatants were harvested, cen-
`trifuged to remove cells and debris, and kept frozen until use
`(-20°C).
`
`699
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`J. Exp. Med. ® The Rockefeller University Press " 0022-1007/91/03/0699/05 $2.00
`Volume 173 March 1991
`699-703
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`ALVOGEN, Exh. 1004, p. 0001
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`TNF-a concentration in the supernatants was
`TNF-a Assay.
`determined with a TNF-a-specific ELISA, specific for the biologi-
`cally active molecule. The reagents were a donation from Dr. Baron
`R. Reed, Genentech, Inc., South San Francisco, CA. Assays were
`performed in 96-well plates (Nunc Immunoplates, Roskilde, Den-
`mark) coated with the affinity-purified rabbit antiTNF-a antibody
`(0.5 ug/ml; 12-16 h; 4°C) and blocked for 2 h at room tempera-
`ture with PBS/0.05% Tween 20 (Sigma Chemical Co., St. Louis,
`MO) containing 5 mg/ml BSA. After washing, 100 P,1 of TNF-a
`standards, samples, and controls were applied to the wells, and the
`plates were incubated for 12-24 h at 4°C. After the incubation,
`plates were washed and a second antibody, horseradish peroxidase
`(HRP)-conjugated mouse monoclonal antiTNF-ac, diluted 1:2,000
`in PBS/BSA/Tween, was applied to the wells and incubated for
`2 h at room temperature. The color reaction was developed with
`the OPD substrate (0.4 mg/ml o-phenylenediamine [Sigma Chem-
`ical Co.] in 24 mM citric acid, 51 mM sodium phosphate, pH 5.0
`[phosphate-citrate buffer; Sigma Chemical Co.], containing 0.012%
`hydrogen peroxide [H202; Fisher Scientific Co., Pittsburgh, PA])
`and absorbance read at 492 nm in an automated ELISA reader (Dy-
`natech Laboratories, Inc., Alexandria, VA).
`ILIl Assays.
`IMO levels were determined using a commercial
`ELISA kit (Cistron Biotechnology, Pine Brook, NJ) according to
`the manufacturer's specifications . 11,10 levels are expressed as pico-
`grams per milliliter of protein.
`IL6 Assay.
`11,6 levels were determined using a biological assay
`as described (9). Proliferation of7TD1 hybridoma cellline specifically
`sensitive to IL6 was measured by colorimetric determination of
`hexosaminidase levels (10), and values for IL6 in the samples were
`obtained by interpolation from a standard curve. 1 U/ml of IL6
`corresponds to the concentration that yields half-maximal growth.
`GM-CSF levels
`Granulocyte/Macrophage CSF (GM-CSF) Assay.
`were determined using a commercial ELISA kit (Genzyme, Boston,
`MA) according to the manufacturer's specifications, and were ex-
`pressed as picograms per milliliter of protein.
`The thalidomide used in this study was
`Thalidomide Inhibition.
`the purified drug (racemic mixture of D [+] and L [-] forms)
`(lot No. JB-I-114 ; Andrulis Research Corporation, Beltsville, MD).
`The compound was shown to be at least 99% pure, melting sharply
`at 270°C (published melting range, 269-271°C) (11). It was then
`diluted in DMSO (Sigma Chemical Co.); further dilutions were
`done in sterile PBS.
`Percentage inhibition ofTNFa secretion was calculated as: 100x
`[1-(TNF-a experimental/TNF-ca control)]; where TNF-a ex-
`perimental represents TNF-ca secretion by stimulated monocytes
`that were cultured in the presence ofthalidomide, and TNF-a con-
`trol represents TNF-ca secretion by stimulated monorytes that were
`cultured in the absence of the drug. Monocytes cultured in medium
`containing equivalent amounts of DMSO in the presence or ab-
`sence ofthe stimulating agent were used as controls for thalidomide-
`treated cells. Neither thalidomide nor DMSO had any effect on
`cell viability or function at the concentrations used.
`Human monocytes were cultured in teflon
`Protein Synthesis.
`beakers in methionine-free RPMI with 10% AB* serum at 37°C
`for 1 h, when 200 pCi/ml [35S]methionine (1,153 Ci/mmol; ICN
`Biomedicals Inc., Irvine, CA) was added to the cultures for the
`next 3 h with or without the stimulating and the suppressive agent.
`At the end of the labeling period, 35S-labeled cells were washed
`twice in ice-cold PBS and lysed directly in 500 P1 lysis solution
`(10 mM Tris-HCI buffer, pH 7.4, 150 NaCl, 1 mM EDTA, and
`1% SDS). Resolving 8% SDS-PAGE was performed overnight. The
`gel was washed, dried, and analyzed by autoradiography at -70°C
`using XAR-5 radiographic film (Kodak, Rochester, NY) with an
`intensifying screen.
`
`700
`
`Results
`Monocytes were enriched from PBMC of normal donors
`and stimulated in vitro for 18-20 h with bacterial LPS and
`mycobacterial products, known agonists ofmonocyte TNF-a
`synthesis and secretion (12, 13). Thalidomide suppressed LPS-
`stimulated TNF-ci production (Fig. 1 A) with a 50% in-
`hibitory concentration (IC5o) of 1-4 P,g/ml, and -90%
`inhibition observed at 10 dug/ml (18-20-h assay). Similar results
`were obtained when PPD and CWRML were used as stimu-
`lants (Fig. 1, B and C, respectively).
`The inhibition of TNF-a secretion by thalidomide was
`dependent upon the state of monocyte stimulation (Table 1).
`Preincubation of unstimulated monocytes with thalidomide,
`followed by removal of the drug before LPS stimulation, did
`not lead to suppression . By comparison, when LPS and
`thalidomide were added simultaneously to the cultures, irre-
`versible suppression occurred, even when the drug was re-
`
`x0a
`
`600
`
`400-
`
`200-
`
`0
`
`E a
`
`THALIDOMIDE (yg/ml)
`Effect of thalidomide on (A) bacterial endotoxin (LPS,
`Figure 1.
`1 jig/ml), (B) PPD, (10 pg/ml), and (C) CWP-ML (10 Wg/ml)-induced
`TNF-a production . Monocyteswere simultaneously incubated with 2 ng/ml
`to 10 Ftg/ml of thalidomide in the culture medium. Control cells were
`cultured in medium alone. A dose-dependent inhibition of TNF-a secre-
`tion by thalidomide was noted. No detectable production of TNF-a pro-
`tein was observed in supernatants ofunstimulated monocytes. Data repre-
`sent mean ± SD of 15 (A), two (B), and one (C) different experiments,
`respectively.
`Thalidomide Selectively Inhibits Tumor Necrosis Factor a Production
`
`ALVOGEN, Exh. 1004, p. 0002
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`Table 1 .
`
`Effect ofPreincubation ofMonocytes with Thalidomide
`
`Time
`
`h
`0-4
`0-4
`None
`0-4
`None
`
`A
`B
`C
`D
`E
`
`Preincubation
`
`Thalidomide
`(4 hg/ml)
`
`LPS
`(1 Wg/ml)
`
`0
`+
`0
`+
`0
`
`0
`0
`0
`+
`0
`
`Time
`
`h
`4-20
`4-20
`0-4
`4-20
`0-20
`
`Incubation
`
`Thalidomide
`(4 gg/ml)
`
`LPS
`(1 Ag/ml)
`
`Percent
`activity
`
`0
`0
`+
`0
`+
`
`+
`+
`+
`+
`+
`
`100
`90 ± 4.6
`48 ± 15
`56 ± 0.5
`52 ± 9.3
`
`Human monocytes cultured in 24-well plates were preincubated with the inhibitory drug with or without the stimulating agent. After 4 h, the
`cultures were washed, medium was replaced, and LPS was added again for the next 16 h. Culture supernatants were recovered at the different periods
`and TNF-a levels determined as described. LPS-induced release of TNF-a by monocytes cultured for 20 h in the absence of thalidomide (A). No
`inhibitory action of thalidomide was detected when the drug was washed away before the addition of the stimulating agent (B). Thalidomide-induced
`inhibition of TNF-a production in the presence of LPS after 4 h of stimulation (C), which perisisted even after the drug was washed away (D).
`Control experiment in which thalidomide was kept in the cultures with the stimulating agent during the whole assay (E). Data represent mean
`± SD of two different experiments.
`
`moved after a few hours (Table 1). Therefore, the thalidomide-
`sensitive reaction(s) occurs only after the LPS induction of
`TNF-a production .
`The inhibition of LPS-stimulated TNF-a secretion by
`thalidomide occurs in a setting in which many other pro-
`teins are being synthesized by both constitutive and induced
`mechanisms (14). Thus, a simple explanation for the effect
`of the drug on TNF-a production could be a suppression
`of overall protein synthesis. Fig. 2 illustrates the effect of
`thalidomide on the pattern and quantity of proteins synthe-
`
`Effect of thalidomide on protein synthesis by human periph-
`Figure 2.
`eral blood monocytes. Electrophoretic analysis of lysates from monocytes
`incubated with [35S]methionine was performed. Cells were stimulated in
`vitro with and without LPS in the presence or absence of thalodomide
`at 1 and 4 Wg/ml. TCA-precipitable radioactivity (10% TCA precipita-
`tion) was measured byliquid scintillation counting. The amount ofradio-
`activity in the pellets is expressed as cpm. x 10- 3 and represents the mean
`of three precipitates with a SD of 10%. Neither total radioactivity nor
`the pattern ofmost ofthe proteinbands in the gel was affected by thalido-
`mide. (Lane 1) Unstimulated cells, 3.3 x 10-2 cpm. in TCA precipitates ;
`(lane 2) cells stimulated with 1 ug/ml LPS, 4.2 x 10 -2 cpm in TCA
`precipitate; (lane 3) cells stimulated with LPS in the presence of 1 Fag/ml
`thalidomide, 4.2 x 10 -2 cpm in TCA precipitate; (lane 4) cells stimu-
`lated with LPS in the presence of4 pg/ml thalidomide, 4.1 x 10-2 cpm
`in TCA precipitate; (lanes S and 6) cells incubated only with thalidomide
`at 1 or 4 F+g/ml, respectively, 3.2 x 10 -2 and 2.8 x 10-2 cpm in TCA
`precipitates, respectively.
`
`701
`
`Sampaio et al.
`
`Levels of different cytokines tested in culture supernatants of
`Figure 3.
`human monocytes stimulated with LPS for 6 h (A-C) or 20 h (D) in the
`presence or absence of4 or 10 P,g/ml ofthalidomide. Data represent mean
`± SD of six different experiments for TNF-a and IL1fl determinations
`and three experiments for IL6 and GM-CSF measurements. About 41.9
`± 14.6% and 52.8 t 14.7% inhibition of TNF-a secretion was found
`in the presence of 4 and 10 FAg/ml of thalidomide, respectively. (Coot)
`Unstimulated cells cultured in medium. No effect on 11,119, I1r6, or GM-
`CSF secretion was detected in these cultures.
`
`ALVOGEN, Exh. 1004, p. 0003
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`

`

`sized after a 3-h pulse of [35S]methionine (legend to Fig. 2).
`The total incorporation of isotope into TCAprecipitable pro-
`teins as well as the intensity of most of the individual bands
`on SDS-PAGE of LPS-triggered monocytes remained un-
`changed after thalidomide treatment.
`Several cytokines are produced by monocytes in response
`to LPS in addition to TNF-ci, including IW/3 and IM (15-16).
`Fig. 3 shows that thalidomide exerts a selective effect by sup-
`pressing only TNF-ot secretionby LPS-stimulated monocytes.
`Whereas 4 /Ag/ml thalidomide suppressed TNF-ci produc-
`tion (41.9% inhibition) (Fig. 3 A), neither 1140 (Fig. 3 B),
`IM (Fig. 3 C), nor GM-CSF production (Fig. 3 D) was
`influenced by the drug. Similar but more extensive selective
`suppression was observed with much higher (up to 20 ug/ml)
`concentrations of thalidomide (not shown).
`
`Discussion
`These experiments demonstrate that thalidomide inhibits
`TNF-a production by human blood monocytes, without
`influencing either general protein synthesis or the expression
`of three other monocyte-derived cytokines. Although cells
`of the mononuclear phagocyte series appear to be the major
`producers of TNF-ci, other cells, in particular, T lympho-
`cytes and NK cells, also synthesize this molecule. Therefore,
`additional experiments clarifying the spectrum of cell types
`sensitive to thalidomide action are now warranted. Experi-
`ments in animals and clinical observations in various inflam-
`matory diseases (17-19) suggest that the production of excess
`TNF-ot is related to a number of toxic manifestations of in-
`
`fection. Fever, cachexia, and general debilitation are usually
`associated with elevated TNF-ci levels, found in cancer (20)
`and a variety of chronic infectious and parasitic diseases, in-
`cluding tuberculosis and the opportunistic infections of the
`acquired immunodeficiency syndrome (21, 22). Therefore, it
`is possible that the use of thalidomide may significantly im-
`prove the quality of life for these patients. In this regard,
`it is important to note that the thalidomide concentrations
`found to be effective in vitro (IC5o at 1-4 jAg/ml) are similar
`to the plasma concentration obtained in man (i.e., up to 1.5
`wg/ml) after the administration of a single oral dose of 150
`mg thalidomide (13). As mentioned, this dose is strikingly
`effective at alleviating the acute symptoms ofENL in leprosy
`patients.
`We must, however, be aware that TNF-ca may play a posi-
`tive role in host resistance, in infections (24) as well as against
`malignancies. Therefore, its suppression could have delete-
`rious effects on host immunity. In this regard, since total
`inhibition ofTNF-a secretion in vitro does not occur at 1-4
`Ag/ml, careful dosing ofthe thalidomide during therapywould
`lead to the persistence of some TNF-ot production. Finally,
`the potent teratogenicity of the drug is well established and
`its use in females of child-bearing age must be carefully con-
`trolled. In this respect, the observation that only one of the
`enantiomers of thalidomide (the S or optical L [-] form)
`displays teratogenic properties (25) is ofinterest. Preliminary
`results obtained in this laboratory suggest that the purified
`nonteratogenic enantiomer R or D (+) isomeric form alone
`is capable of suppressing TNF-a production, similar to the
`racemic mixture.
`
`We thank A. R. de Moura for the graphic work and M. Garcia for efficient secretarial help. Thanks are
`due to P. A. Frindt for technical assistance and to J. Ming for performing the IIr6 assay measurements
`and to W. Swiggard for helpful discussion.
`These studies were supported in part by U.S. Public Health Service grant AI-22616. E. P. Sampaio is
`a Villares Fellow.
`Address correspondence to Gilla Kaplan, Laboratory of Cellular Physiology and Immunology, The Rocke-
`feller University, 1230 York Avenue, New York, NY 10021. Ruth Galilly's present address is the Lauten-
`berg Center for General and Tumor Immunology, The Hebrew University, Hadassah Medical School,
`Jerusalem 91010, Israel.
`
`Received for publication 21 November 1990.
`
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`703
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`Sampaio et al.
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`ALVOGEN, Exh. 1004, p. 0005
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