- Email: [email protected]
Tumor necrosis factor α induction in human monocytes
TUMOR
NECROSIS FACTOR a INDUCTION HUMAN MONOCYTES
IN
Natalia 1. Misuno,’
Oleg A. Osipovich,’ Andrew B. Sudarikov,2 Tatyana S. Kolesnikova,’ Gregory L. Idelson,3 Alexander V. Panyutich,’ Nikolai N. Voitenok2
The present study was undertaken to assess the presence of tumor necrosis factor (TNF)-(U mRNA and protein in circulating human blood monocytes and to study the TNF-a gene expression in human monocytes isolated by continuous Percoll gradient fractionation. The technique of RNA isolation directly from the blood samples was used to study TNF-cx mRNA expression in circulating human blood leukocytes. It was shown that human blood leukocytes of healthy donors contained no presynthesized pool of TNF-(Y mRNA as well as no TNF-a protein. It was found that early pretreatment with cycloheximide interfers with TNF-a mRNA induction by Staphylococcus aureus. o 1990 by W.B. Saunders Company.
Tumor necrosis factor 01 (TNF-a) is the macrophage cytotoxin and one of the major inflammatory monokines.1’2 TNF-LU is strongly implicated in the pathogenesis of septic shock. 2*3 In an animal endotoxininduced shock model TNF-or elaboration occurs within minutes of intravenous endotoxin administration.lm3 The existence of a presynthesized pool of TNF-cx mRNA in macrophages in vivo is suspected.274Detectable levels of TNF-a! mRNA were found in thioglycolate-elicited mouse peritoneal macrophages4 and in the organs of naive rats in vivo.5 Recently it was shown that induction of TNF-(U synthesis in human blood monocytes in vitro is resistant to the action of actinomycin D (AcD).~,’ In contrast, induction of lymphotoxin (LT) in human lymphocyte culture was blocked by AcD.~ It was suggested that human blood monocytes also contain presynthesized pools of TNF-or mRNA.6*7 In the studies described here, we have used freshly Percoll-separated blood monocytes to assess the presence of TNF-ar mRNA and TNF-a! protein in circulat-
‘Byelorussian Institute of Hematology and Blood Transfusion, 223059 Minsk, USSR. *Institute of Experimental Hematology and Biotechnology, All Union Scientific Center of Hematology, 125 167 Moscow, USSR. ‘The Lautenberg Center for General and Tumor Immunology, P.O. Box 1172, Jerusalem 91010, Israel. *To whom correspondence should be addressed at Inst. of Experimental Hematology & Biotechnology, Novozykovsky pr. 4a, 125167 Moscow, USSR. 0 1990 by W.B. Saunders Company. 1043-4666/90/0206-0002$05.00/O KEY WORDS: Monocyte activation/mRNA
464
isolation/TNF-a
ing human blood monocytes and to study TNF-a gene expression in human monocytes that were not preactivated by adherence separation. Taking into account possible low stability of TNF-a! mRNA in resting cells and the possibility of TNF-a mRNA synthesis during monocyte isolation, we apply the technique of RNA isolation directly from blood samples. We have shown that circulating blood monocytes of healthy donors contain no presynthesized pool of TNF-ol mRNA as well as no preformed TNF-a! protein. Differences in the cycloheximide (Chx) effect on TNF-cu mRNA accumulation were observed in resting and strongly activated monocytes. We suggest that TNF-a mRNA synthesis may be an attribute of macrophage differentiation of monocytic cells. RESULTS Measurement of TNF-cw in Freshly Isolated Cultured Monocytes
and
Circulating human blood monocytes carry a lot of intracytoplasmic and membrane-bound proteins. We studied the possibility of storage of presynthesized TNF-(r in circulating monocytes. It was shown (Table 1) that freshly isolated monocytes contained no TNF-cu detectable by ELISA, or at least less then 20 picograms of TNF-ol per 2 x lo7 cells (fewer than 40 molecules per monocyte). Similar results were obtained in experiments utilizing either monoclonal antibodies to distinct TNF-(Y epitopes (Table 1) or polyclonal antibodies against TNF-LX (not shown). Activation of monocytes by Staphylococcus aureus Cowan 1 (SAC) caused the appearance
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TNF-cr induction in human monocytes / 465 TABLE 1. ELISA for the presence of TNF in freshly isolated and in vitro incubated human monocytes. (nanograms Treatment Experiment
TNF per 2 x 10’ cells)
Intracellular*
secretedt
did not influence monocyte viability (not shown). Thus, observed inhibition of TNF-ol gene expression was not due to cytotoxic action of Chx in monocyte culture. When monocytes were isolated from the blood portion containing AcD at 10 yg/mL and further
1
1. Freshly isolated SAC, 2 hr. SAC, 18 hr. Experiment 2 1. Freshly isolated 2. SAC, 2 hr. 3. SAC, 18 hr. 4. SAC Chx, 18 hr. 2. 3.
10.02
-
§
26.72 229.7
0.34
-
-co.02 0.52
25.92 508.5 19.19
1.10 0.05
*Intracellular: total amount of TNF in lysatesof 2 x lo7 cells. tSecreted: total amount of TNF secreted by 2 x 10’ monocytes medium. §No detection was performed at that point.
of intracellular TNF-(u, but did not lead to significant intracellular accumulation of cytotoxin compared to the amount of secreted TNF-ol (Table 1). The amount of TNF-a! elaborated during 18 hr of SAC activation was about 500 nanograms per 2 x lo7 cells (or about 1,OOO,OOO molecules of TNF-cu per monocyte). Cycloheximide inhibited production of both intracellular and secreted TNF-a. TNF-(r mRNA Detection Cultured Monocytes
1
2
1
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3
4
5
6
7
into the culture
in Freshly Isolated
and
The TNF-a mRNA level was undetectable by conventional Northern analysis in freshly Percollisolated human blood monocytes (Fig. 1). To stabilize possible stored mRNA in blood monocytes and to prevent the induction of new mRNA during cell separation, portions of the same donor blood were collected into tubes containing Chx (10 pg/mL) or AcD (10 a/mL). ‘3’ TNF-a mRNA was not detected in freshly Percoll-isolated monocytes from Chx- or AcD-treated blood. Traces of TNF-a mRNA were found in unstimulated monocytes incubated for 3 hr. The level of TNF-ol mRNA in unstimulated human monocytes was significantly increased when monocytes were isolated and cultured in the presence of Chx without additional stimulation (Fig. 1). Addition of SAC to purified monocytes in vitro resulted in accumulation of large quantities of TNF-a mRNA. When monocytes were isolated and cultured in the presence of Chx, the level of TNF-LU mRNA induced by SAC was decreased about two-fold (data from three experiments) or was not changed. Similar inhibition of SAC-induced mRNA accumulation was detected when monocytes were isolated from the blood portion without Chx and were incubated with Chx only in SAC-activated cell cultures for 3 hr and 5 hr. Chx at 10 wg/mL added for 3 or 5 hr of incubation
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Figure 1. Northern blot analysis of TNF-a transcripts in isolated human monocytes treated with SAC (0.001%) and/or Chx (10 pg/mL) for the indicated time. Total cellular RNA (15 pg/lane) was extracted and analyzed as described in the Materials and Methods section. (A) Lane 1, Freshly isolated monocytes. 2, Freshly isolated monocytes (AcD was added to the blood sample before monocyte isolation). 3, Freshly isolated monocytes (Chx was added to the blood sample before monocyte isolation). 4, 3 hr of monocyte incubation. 5, 3 hr of monocyte incubation (Chx was added to the blood sample before monocyte isolation). 6, 3 hr of monocyte incubation with SAC. 7, 3 hr of monocyte incubation with SAC and Chx (Chx was added to the blood sample before monocyte isolation). (B) Lane 1, Freshly isolated monocytes. 2, Freshly isolated monocytes (AcD was added to the blood sample before monocyte isolation). 3, Freshly isolated monocytes (Chx and AcD were added to the blood sample before monocyte isolation). 4,3 hr of monocyte incubation with SAC (AcD and Chx were added to the blood sample before monocyte isolation). 5, 3 hr of monocyte incubation with SAC. 6, 3 hr of monocyte incubation with SAC (AcD was added to the blood sample before monocyte isolation). 7, 12 hr of monocyte incubation with SAC. (C) Lane 1, Freshly isolated monocytes. 2, 3 hr of monocyte incubation. 3, 5 hr of monocyte incubation. 4, 3 hr of monocyte incubation with SAC. 5, 5 hr of monocyte incubation with SAC. 6, 3 hr of monocyte incubation with SAC and Chx. 7,5 hr of monocyte incubation with SAC and Chx.
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activated by SAC, accumulation of TNF-a! mRNA was decreased compared to the level of TNF-a mRNA in SAC-stimulated monocytes from the blood portion without AcD, but was not blocked completely. Estimation Samples
of TNF-a
mRNA in Whole Blood
Taking into account possible degradation of preformed mRNA in blood monocytes during the isolation procedure, we have measured the level of TNF-ar mRNA directly in fresh unfractionated blood samples. TNF-a! mRNA was undetectable in RNA samples isolated directly from fresh unfractionated blood (Fig. 2). Incubation of the blood for 3 hr at 37% caused accumulation of detectable TNF-a mRNA (Fig. 2). Addition of Chx to the whole blood resulted in enhancement of TNF-cx mRNA accumulation in the blood samples cultured for 3 hr without additional stimulation (Fig. 2). Incubation of the blood for 3 hr at 37% in the presence of SAC induced large quantities of TNF-a! transcripts. In SACactivated blood samples, the addition of Chx resulted in enhancement of TNF-a mRNA accumulation. In the presence of 10 pg/mL of AcD no TNF-LU mRNA was detected in the blood sample incubated for 3 hr at 37% Incubation of the whole blood in vitro for 3 hr at 37°C with SAC was accompanied by production of biologically active TNF-cr. The background level of TNF-(Y cytotoxic activity in blood incubated for 3 hr without activation was less than 4 RU/mL, in SAC activated blood it was 248 * 38 reference units (RU)/mL (mean of 4 experiments; SAC-induced activity was completely blocked by neutralizing anti-TNF-a monoclonal antibodies). In the presence of Chx, accumulation
A
1
2
Figure
2.
3
8
4
Northern
5
12345
blot analysis
of TNF-a
transcripts
in the whole
Total RNA (25 fig/lane) was isolated from whole blood (see the Materials and Methods section) and treated with SAC (O.OOl%), Chx (10 Gg/mL), or AcD (10 pg/mL) for the indicated time. (A) Lane 1, Fresh blood, 0 hr. 2, 3 hr of blood incubation. 3, 3 hr of blood incubation with SAC. 4,3 hr of blood incubation with AcD. 5, 3 hr of blood incubation with SAC and Chx. (B) Lane 1, Fresh blood, 0 hr. 2, 3 hr of blood incubation. 3,3 hr of blood incubation with SAC. 4,3 hr of blood incubation with SAC and Chx. 5, 3 hr of blood incubation with Chx.
Vol. 2, No. 6 (November 1990: 464-469)
of TNF-ol induced by SAC was abrogated (less than 4 RU/mL in the experiments mentioned above). DISCUSSION The present study was undertaken because of the data indicating that induction of TNF-(U synthesis in human monocyte culture and in whole blood exposed to lipopolysaccharide (LPS) in vitro6,7 cannot be prevented by AcD treatment. It was suggested that TNF-cr mRNA can be presynthesized in human blood monocytes.6,7 Another explanation for these results was the possibility of storage of presynthesized pro-TNF-a within circulating monocytes. It was previously demonstrated that human blood monocytes isolated from the human blood mononuclear cell fraction by adherence to plastic contained no TNF-a! mRNA detectable by conventional Northern analysis.” However, it could not be excluded that dormant mRNA containing AU rich sequences in the 3’-untranslated region could be very unstable in the absence of appropriate cell activation signals1~16-‘8and could be degraded during monocyte isolation by plastic adherence from blood mononuclear cell fractions. Using continuous Percoll gradient centrifugation and cold separation media for purification of human monocytes, we detected no TNF-a mRNA in RNA samples from freshly isolated monocytes. The same result was obtained when AcD and Chx were added to the blood samples immediately after venepuncture. In addition, presynthesized TNF-cu protein was not detected by ELISA in lysates of freshly isolated monocytes using both monoclonal and polyclonal antibodies, indicating that TNF-(r protein was also not stored in circulating human monocytes. To exclude any possibility of hypothetical dormant TNF-cr mRNA destruction during the monocyte separation procedure we have established a simple technique of RNA isolation from whole blood. The whole blood samples were immediately lysed after venepuncture by the buffer inactivating RNAse activity. Using this approach we detected no TNF-cu mRNA in RNA samples isolated from fresh donor blood. Efficiency of this technique for RNA isolation was proved by the demonstration of TNF-(U mRNA in RNA samples isolated from the whole blood exposed to SAC for 3 hr. Thus, the results of the present study show that circulating human blood monocytes contain no stored pool of TNF-ol mRNA or protein and imply that induction of TNF-ol synthesis in blood monocytes requires TNF-a! gene transcription activation and translation. It was demonstrated that the whole blood as well as Percoll-isolated monocytes incubated alone for 3 hr can produce low but detectable amounts of TNF-a mRNA. “Spontaneous” TNF-a! mRNA accumulation was in-
TNF-a inductionin human monocytes/ 467
creased many-fold in the presence of Chx. It was unforeseen to find that the presence of Chx during monocyte isolation and in the monocyte culture activated by SAC caused no “superinduction” of TNF-a mRNA or caused lowering of TNF-a mRNA level. The data suggest that early pretreatment of monocytes with Chx might interfere with TNF-cr mRNA induction in purified monocyte culture under the influence of the potent activating signal provided by SAC. These data imply that different mechanisms can operate in initiation of TNF-a gene transcription in resting, cultured, and activated monocytes. These results seem to conflict with the previous studies that demonstrated “superinduction” of TNF-a mRNA accumulation by Chx in human monocytes isolated by long-time plastic adherence (i.e., macrophages)15 and in mouse peritoneal macrophages elicited by thioglycolate injection.ig This discrepancy might be due to the use of non-activated blood monocytes freshly isolated by Percoll centrifugation in our experiments. In contrast, addition of Chx to the whole blood samples activated by SAC caused enhancement of TNF-a mRNA accumulation. It can be suggested that differential action of Chx on TNF-a mRNA accumulation in Percoll-isolated monocytes and in the whole blood exposed to SAC might be due to the presence of other leukocytes in the whole blood, which also could accumulate TNF-(U mRNA. The synthesis of TNF-a mRNA by human polymorphonuclear leukocytes,” T cells,21 and NK cells2’ has been described. In the present experiments it was shown that the protocol of RNA synthesis inhibition by AcD used in
previous reports6v7 was insufficient to block TNF-a mRNA induction. According to our preliminary data, it was due to the relative resistance of monocyte RNA
synthesis to the action of AcD. It seems that the absence of TNF-a mRNA in the samples of whole blood demonstrates that there are no
pools of TNF-(U mRNA sufficient for detectable production of TNF-a stored in circulating human blood monocytes. It can be hypothesized that TNF-a mRNA
water at 1 mg/mL and stored at -2OOC. Cycloheximide (Chx) (Calbiochem, La Jolla, CA) was dissolved in sterile water at 1 mg/mL and stored at -20°C. RPMI-1640 was purchased from Gibco. Neutralizing monoclonal antibodies
(MAb) (IgGl) against human TNF-o( were produced by 3C7N hybridoma cells grown in ascites.’ Percoll was from Pharmacia, Uppsala. Continuous Percoll gradient separation was performed as described previously.” A sterile solution of heparin for injection (Richter, Budapest) was used for blood stabilization at 25 IU/mL.
Isolation of Monocytes Mononuclear leukocyteswere isolated from fresh heparinized blood of healthy volunteers using a standard Ficoll/
Hypaque (F/H) protocol at +4”C. In some experiments the portion of the donor blood was collected from the venepuncture system directly into bottles that contained the amount of AcD sufficient to achievea final concentration of 10 pg/mL and was further separated by F/H. Cooled mononuclear leukocytes from F/H were fractionated on a continuous Percoll gradient at +4°C.6,io Isolated monocytes were more than 80% naphtyl acetate esterase-positive (NAE+) (NAE kit from Sigma).
Induction of TNF-cwin Monocyte Cultures Monocytes were washed in cold medium, resuspended in 5% FCS-RPM1 at 1.5 x lo6 to 2.0 x lo6 cells/ml and incubated in 50- or loo-mm polystyrene culture dishes (Flow Laboratories). To induce TNF-or synthesis,sterile SAC suspension was added to the monocyte cultures at a final concentration of 0.001% (v/v). Supernatants were decanted and tested for TNF-(r activity. Cells were harvested with a “rubber policeman” and lysed (seebelow). To inhibit protein synthesis, Chx was added to monocyte cultures at a concentration of 10 pg/mL. To inhibit protein or mRNA synthesis very early before cell isolation procedure, portions of blood of the same donor were collected directly into the tubes that contained Chx (10 pg/mL) or AcD (10 pg/mL). Mononuclear cells were further isolated as described above. When monocytes were isolated from Chx-treated blood, all solutions contained the indicated concentration of the drug.
Whole Blood Incubation in Vitro
analysis in human diseases.
Donor blood was collected directly into 50-mL polypropylene tubes (Flow Laboratories) that contained SAC (for a final concentration of 0.001% v/v), AcD (10 pg/mL), or Chx (10 pg/mL). The tubes were incubated at 37°C in a water bath for 3 hr. A portion of incubated blood was immediately used for RNA extraction, the other portion was sedimented, and the plasma was utilized for detection of TNF-cu.
MATERIALS
TNF-a Bioassay
synthesis may be an attribute of macrophage differentiation of monocytes and is incompatible with the capacity of blood monocytes to circulate. The technique of
TNF-a mRNA isolation from the whole blood samples described here might be useful for TNF-a mRNA
AND
METHODS
Reagents Staphylococcus aureus Cowan 1 (SAC) was prepared as described* at a concentration of 5% in sterile saline and stored at +4”C. Actinomycin D (AcD) (Serva, Heidelberg) was dissolved in ethanol at 1 mg/mL and 5 mg/mL or in sterile
Cytotoxic activity of TNF-(r was measured by conventional L-929 cell cytotoxicity photometric assay in the presence of 1 pg/mL of AcD.” A unit of cytotoxin was defined as the reciprocal of the dilution of cytotoxin preparation causing 50% cytotoxicity and was expressed as RU according to titration against a TNF-o bioactivity standard (25 pg of
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human TNF-(U = 1 RU) provided by the National Institute for Biological Standards and Control, UK. For the neutralization assay,serial dilutions of test sampleswere incubated with an excessof neutralizing monoclonal antibodies (MAb) against human TNF-a! for 1 hr at 37°C before the cytotoxicity assay.
on TNF-a mRNA accumulation in human leukocytes and Dr. Sergei Nedospasov for kindly providing genomic DNA of human TNF-a, which was used for the construction of the TNF-a probe in M 13mpl9 phage.
ELISA Analysis
REFERENCES
Collected cells were lysed in ice-cold phosphate buffer containing 1% NP-40 and protease inhibitors: 1 mM phenylmethane-sulfonylfluoride (PMSF), 1 mM N-ethylmaleimide (NEM), 1 mM diethylenetriamine pentaacetic acid (DTPA). After 3 x 10 set of ultrasonic treatment lysates were centrifuged and supernatants were tested. Monoclonal anti-TNF-cu (3C7N at a concentration of 0.01 mg/mL of sodiumcarbonate buffer, pH 9.5) was added to a 96-well microtest plate and incubated overnight at 4°C. The plates were washed 5 times with PBS-O.05% Tween-20 (PBS-T), and TNF-a! sampleswere added and incubated for 40 min (shaking). After five washings with PBS-T, rabbit IgG against human recombinant TNF-a was added for 40 min. Then the plates were washed 5 times with PBS-T and peroxidase-conjugated goat anti-rabbit Ig in PBS-T with 10% sheep serum and 0.1 mg/mL of mouse Ig was added and the incubation was continued for 40 min. After washing with PBS-T, 0.1 mL of 0.6 mg/mL o-phenylendiamine, HCl and 1 mM H,O, in 0.02 M sodium citrate buffer were added and incubated for 30 min. After addition of 50% H,SO,, the optical density of the samples was measured at 492 nm. The concentration of TNF-(U was calculated against a recombinant human TNF-(U ELISA titration.
Northern Blot Analysis RNA from isolated monocytes was extracted by a conventional procedure.‘* To extract RNA from unfractionated blood, the samples of blood (5 mL) were mixed with an equal volume of hot (60°C) lysis solution containing 8 M guanidine thiocyanate, 50 mM sodium citrate, 1% sarcosyl, and 200 mM 2-mercaptoethanol. To minimize the influence of in vitro manipulations, blood samples representing intact (zero time) circulating monocytes were collected from the venepuncture system directly into tubes containing equal volumes of hot lysis solution. Starting at this point isolation of RNA was done as described in reference 12. Fifteen to twenty-five micrograms of total RNA was subjected to formaldehyde-MOPS denaturing electrophoresis (as described in reference 13). Sample loading and RNA integrity were controlled by ethidium bromide fluorescencephotography of gels (data not shown). As a probe for TNF-(U mRNA we used a TNF-ol gene fragment (positions 5793 to 5928 according to reference 14) inserted in M13mp19 phage DNA and labeled by the primer extension method to the specific activity of lo9 cpm/Fg. All manipulations of RNA, blots, and DNA probes were done essentially as described in reference 13. Autoradiographs were developed after overnight exposure with an intensifying screen. Acknowledgments We would like to thank Dr. Daniela Mannel for kindly providing data concerning the influence of SAC
1. Beutler B, Cerami A (1986) Cachectin and tumor necrosis factor as two sides of the same biological coin. Nature 320:584-588. 2. Beutler B, Cerami A (1990) Cachectin (tumor necrosis factor): an endogenous mediator of shock and inflammatory response. In Oppenheim JJ, Shervach EM (eds) Immunophysiology, The Role of Cells and Cytokines in Immunity and Inflammatory, Oxford University Press, New York, Oxford, pp 226-237. 3. Mathison JC, Wolfson E, Ulevitch RJ (1988) Participation of tumor necrosis factor in the mediation of gram negative bacterial lipopolysaccharide-induced injury in rabbits. J Clin Invest 81:19251937. 4. Beutler B, Krochin N, Milsark IW, Luedke C, Cerami A (1986) Control of cachectin (tumor necrosis factor) synthesis: mechanisms of endotoxin resistance. Science 232:977-980. 5. Ulich TR, Guo K, de1 Castillo J (1989) Endotoxin-induced cytokine gene expression in vivo. 1. Expression of tumor necrosis factor mRNA in visceral organs under physiological conditions and during endotoxemia. American J Path 134:11-14. 6. Voitenok NN, Misuno NI, Panyutich AV, Kolesnikova TS (1989) Induction of tumor necrosis factor synthesis in human monocytes treated by transcriptional inhibitors. Immunol Lett 20:77-82. 7. Hofsli E, Lamvik J, Nissen-Meyer J (1988) Evidence that tumor necrosis factor (TNF-ol) is not constitutively present in vivo. Stand J Immunol28:435-441. 8. Kessler SW (1975) Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol 115:1617-1624. 9. Panyutich AV, Karkanitsa LV, Komarovskaya ME, Nevmerzhitskaya SI, Voitenok NN (1988) Neutralizing antibodies against tumor necrosis factor used for evaluating activity of colonystimulating hemopoietic factors in cultures containing macrophages. Hematol and Transfusiol (in Russian) 12:21-24. 10. Gmelig-Meyling F, Waldmann TA (1980) Separation of human blood monocytes and lymphocytes on a continuous Percoll gradient. J Immunol Meth 33:1-9. 11. Fisch H, Gifford JE (1983) A photometric and plaque assay for macrophage mediated tumor cell cytotoxicity. J Immunol Meth 2:311-325. 12. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chlorophorm extraction. Anal Biochem 162: 156- 159. 13. Maniatis T, Fritsch EE, Sambrook J (1982) Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 14. Nedospasov SA, Shakhov AN, Turetskaya RL, Mett VA, Azizov MM, Georgiev GP, Korobko VG, Dobrynin VN, Filippov SA, Bystrov NS, Boldyreva EF, Chuvpilo SA, Chumakov AM, Shingarova LN, Ovchinnikov YA (1986) Tandem arrangement of genes coding for tumor necrosis factor (TNF-o() and lymphotoxin (TNF-p) in the human genome. Cold Spring Harbor Symp Quant Biol51:61 l-624. 15. Sariban E, Imamura K, Luebbers R, Kufe D (1988) Transcriptional and posttranscriptional regulation of tumor necrosis factor gene expression in human monocytes. J Clin Invest 81:15061510. 16. Shaw G, Kamen R (1986) A conserved AU sequence from 3’untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46:659-667.
TNF-cu induction in human monocytes / 469 17. Strickland S, Huarte J, Belin D, Vassali A, Rickeles RJ, Vassali J-D (1988) Antisense RNA directed against the 3’ noncoding region prevents dormant mRNA activation in mouse oocytes. Science 241:680-684. 18. Ross J (1989) The turnover of messenger RNA. Scientific American 4:16-23. 19. Collart MA, Belin D, Vassali J-D, de Kossodo S, Vassali P (1986) Interferon enhances macrophage transcription of the tumor necrosis factor/cachectin, interleukin 1, and urokinase genes, which are controlled by short-lived repressors. J Exp Med 164:2113-2118.
20. Lindemann A, Riedel D, Oster W, Ziegler-Heitbrock HWL, Mertelsmann R, Herrmann F (1989) Granulocyte-macrophage colonystimulating factor induces cytokine secretion by human polymorphonuclear leukocytes. J Clin Invest 83:1308-1312. 21. Lidsten T, June CH, Ledbetter GA, Stella G, Tompson CB (1989) Regulation of lymphokine messenger RNA stability by surfacemediated T-cell activation pathway. Science 244:339-343. 22. Ortaldo JR, Mason LH, Matieson BJ, Liang SM, Flick DA, Herberman RB (1986) Mediation of natural cytotoxic activity by tumor necrosis factor. Nature 321:700-704.
NECROSIS FACTOR a INDUCTION HUMAN MONOCYTES
IN
Natalia 1. Misuno,’
Oleg A. Osipovich,’ Andrew B. Sudarikov,2 Tatyana S. Kolesnikova,’ Gregory L. Idelson,3 Alexander V. Panyutich,’ Nikolai N. Voitenok2
The present study was undertaken to assess the presence of tumor necrosis factor (TNF)-(U mRNA and protein in circulating human blood monocytes and to study the TNF-a gene expression in human monocytes isolated by continuous Percoll gradient fractionation. The technique of RNA isolation directly from the blood samples was used to study TNF-cx mRNA expression in circulating human blood leukocytes. It was shown that human blood leukocytes of healthy donors contained no presynthesized pool of TNF-(Y mRNA as well as no TNF-a protein. It was found that early pretreatment with cycloheximide interfers with TNF-a mRNA induction by Staphylococcus aureus. o 1990 by W.B. Saunders Company.
Tumor necrosis factor 01 (TNF-a) is the macrophage cytotoxin and one of the major inflammatory monokines.1’2 TNF-LU is strongly implicated in the pathogenesis of septic shock. 2*3 In an animal endotoxininduced shock model TNF-or elaboration occurs within minutes of intravenous endotoxin administration.lm3 The existence of a presynthesized pool of TNF-cx mRNA in macrophages in vivo is suspected.274Detectable levels of TNF-a! mRNA were found in thioglycolate-elicited mouse peritoneal macrophages4 and in the organs of naive rats in vivo.5 Recently it was shown that induction of TNF-(U synthesis in human blood monocytes in vitro is resistant to the action of actinomycin D (AcD).~,’ In contrast, induction of lymphotoxin (LT) in human lymphocyte culture was blocked by AcD.~ It was suggested that human blood monocytes also contain presynthesized pools of TNF-or mRNA.6*7 In the studies described here, we have used freshly Percoll-separated blood monocytes to assess the presence of TNF-ar mRNA and TNF-a! protein in circulat-
‘Byelorussian Institute of Hematology and Blood Transfusion, 223059 Minsk, USSR. *Institute of Experimental Hematology and Biotechnology, All Union Scientific Center of Hematology, 125 167 Moscow, USSR. ‘The Lautenberg Center for General and Tumor Immunology, P.O. Box 1172, Jerusalem 91010, Israel. *To whom correspondence should be addressed at Inst. of Experimental Hematology & Biotechnology, Novozykovsky pr. 4a, 125167 Moscow, USSR. 0 1990 by W.B. Saunders Company. 1043-4666/90/0206-0002$05.00/O KEY WORDS: Monocyte activation/mRNA
464
isolation/TNF-a
ing human blood monocytes and to study TNF-a gene expression in human monocytes that were not preactivated by adherence separation. Taking into account possible low stability of TNF-a! mRNA in resting cells and the possibility of TNF-a mRNA synthesis during monocyte isolation, we apply the technique of RNA isolation directly from blood samples. We have shown that circulating blood monocytes of healthy donors contain no presynthesized pool of TNF-ol mRNA as well as no preformed TNF-a! protein. Differences in the cycloheximide (Chx) effect on TNF-cu mRNA accumulation were observed in resting and strongly activated monocytes. We suggest that TNF-a mRNA synthesis may be an attribute of macrophage differentiation of monocytic cells. RESULTS Measurement of TNF-cw in Freshly Isolated Cultured Monocytes
and
Circulating human blood monocytes carry a lot of intracytoplasmic and membrane-bound proteins. We studied the possibility of storage of presynthesized TNF-(r in circulating monocytes. It was shown (Table 1) that freshly isolated monocytes contained no TNF-cu detectable by ELISA, or at least less then 20 picograms of TNF-ol per 2 x lo7 cells (fewer than 40 molecules per monocyte). Similar results were obtained in experiments utilizing either monoclonal antibodies to distinct TNF-(Y epitopes (Table 1) or polyclonal antibodies against TNF-LX (not shown). Activation of monocytes by Staphylococcus aureus Cowan 1 (SAC) caused the appearance
CYTOKINE,
Vol. 2, No. 6 (November), 1990: pp 464-469
TNF-cr induction in human monocytes / 465 TABLE 1. ELISA for the presence of TNF in freshly isolated and in vitro incubated human monocytes. (nanograms Treatment Experiment
TNF per 2 x 10’ cells)
Intracellular*
secretedt
did not influence monocyte viability (not shown). Thus, observed inhibition of TNF-ol gene expression was not due to cytotoxic action of Chx in monocyte culture. When monocytes were isolated from the blood portion containing AcD at 10 yg/mL and further
1
1. Freshly isolated SAC, 2 hr. SAC, 18 hr. Experiment 2 1. Freshly isolated 2. SAC, 2 hr. 3. SAC, 18 hr. 4. SAC Chx, 18 hr. 2. 3.
10.02
-
§
26.72 229.7
0.34
-
-co.02 0.52
25.92 508.5 19.19
1.10 0.05
*Intracellular: total amount of TNF in lysatesof 2 x lo7 cells. tSecreted: total amount of TNF secreted by 2 x 10’ monocytes medium. §No detection was performed at that point.
of intracellular TNF-(u, but did not lead to significant intracellular accumulation of cytotoxin compared to the amount of secreted TNF-ol (Table 1). The amount of TNF-a! elaborated during 18 hr of SAC activation was about 500 nanograms per 2 x lo7 cells (or about 1,OOO,OOO molecules of TNF-cu per monocyte). Cycloheximide inhibited production of both intracellular and secreted TNF-a. TNF-(r mRNA Detection Cultured Monocytes
1
2
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5
6
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into the culture
in Freshly Isolated
and
The TNF-a mRNA level was undetectable by conventional Northern analysis in freshly Percollisolated human blood monocytes (Fig. 1). To stabilize possible stored mRNA in blood monocytes and to prevent the induction of new mRNA during cell separation, portions of the same donor blood were collected into tubes containing Chx (10 pg/mL) or AcD (10 a/mL). ‘3’ TNF-a mRNA was not detected in freshly Percoll-isolated monocytes from Chx- or AcD-treated blood. Traces of TNF-a mRNA were found in unstimulated monocytes incubated for 3 hr. The level of TNF-ol mRNA in unstimulated human monocytes was significantly increased when monocytes were isolated and cultured in the presence of Chx without additional stimulation (Fig. 1). Addition of SAC to purified monocytes in vitro resulted in accumulation of large quantities of TNF-a mRNA. When monocytes were isolated and cultured in the presence of Chx, the level of TNF-LU mRNA induced by SAC was decreased about two-fold (data from three experiments) or was not changed. Similar inhibition of SAC-induced mRNA accumulation was detected when monocytes were isolated from the blood portion without Chx and were incubated with Chx only in SAC-activated cell cultures for 3 hr and 5 hr. Chx at 10 wg/mL added for 3 or 5 hr of incubation
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Figure 1. Northern blot analysis of TNF-a transcripts in isolated human monocytes treated with SAC (0.001%) and/or Chx (10 pg/mL) for the indicated time. Total cellular RNA (15 pg/lane) was extracted and analyzed as described in the Materials and Methods section. (A) Lane 1, Freshly isolated monocytes. 2, Freshly isolated monocytes (AcD was added to the blood sample before monocyte isolation). 3, Freshly isolated monocytes (Chx was added to the blood sample before monocyte isolation). 4, 3 hr of monocyte incubation. 5, 3 hr of monocyte incubation (Chx was added to the blood sample before monocyte isolation). 6, 3 hr of monocyte incubation with SAC. 7, 3 hr of monocyte incubation with SAC and Chx (Chx was added to the blood sample before monocyte isolation). (B) Lane 1, Freshly isolated monocytes. 2, Freshly isolated monocytes (AcD was added to the blood sample before monocyte isolation). 3, Freshly isolated monocytes (Chx and AcD were added to the blood sample before monocyte isolation). 4,3 hr of monocyte incubation with SAC (AcD and Chx were added to the blood sample before monocyte isolation). 5, 3 hr of monocyte incubation with SAC. 6, 3 hr of monocyte incubation with SAC (AcD was added to the blood sample before monocyte isolation). 7, 12 hr of monocyte incubation with SAC. (C) Lane 1, Freshly isolated monocytes. 2, 3 hr of monocyte incubation. 3, 5 hr of monocyte incubation. 4, 3 hr of monocyte incubation with SAC. 5, 5 hr of monocyte incubation with SAC. 6, 3 hr of monocyte incubation with SAC and Chx. 7,5 hr of monocyte incubation with SAC and Chx.
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activated by SAC, accumulation of TNF-a! mRNA was decreased compared to the level of TNF-a mRNA in SAC-stimulated monocytes from the blood portion without AcD, but was not blocked completely. Estimation Samples
of TNF-a
mRNA in Whole Blood
Taking into account possible degradation of preformed mRNA in blood monocytes during the isolation procedure, we have measured the level of TNF-ar mRNA directly in fresh unfractionated blood samples. TNF-a! mRNA was undetectable in RNA samples isolated directly from fresh unfractionated blood (Fig. 2). Incubation of the blood for 3 hr at 37% caused accumulation of detectable TNF-a mRNA (Fig. 2). Addition of Chx to the whole blood resulted in enhancement of TNF-cx mRNA accumulation in the blood samples cultured for 3 hr without additional stimulation (Fig. 2). Incubation of the blood for 3 hr at 37% in the presence of SAC induced large quantities of TNF-a! transcripts. In SACactivated blood samples, the addition of Chx resulted in enhancement of TNF-a mRNA accumulation. In the presence of 10 pg/mL of AcD no TNF-LU mRNA was detected in the blood sample incubated for 3 hr at 37% Incubation of the whole blood in vitro for 3 hr at 37°C with SAC was accompanied by production of biologically active TNF-cr. The background level of TNF-(Y cytotoxic activity in blood incubated for 3 hr without activation was less than 4 RU/mL, in SAC activated blood it was 248 * 38 reference units (RU)/mL (mean of 4 experiments; SAC-induced activity was completely blocked by neutralizing anti-TNF-a monoclonal antibodies). In the presence of Chx, accumulation
A
1
2
Figure
2.
3
8
4
Northern
5
12345
blot analysis
of TNF-a
transcripts
in the whole
Total RNA (25 fig/lane) was isolated from whole blood (see the Materials and Methods section) and treated with SAC (O.OOl%), Chx (10 Gg/mL), or AcD (10 pg/mL) for the indicated time. (A) Lane 1, Fresh blood, 0 hr. 2, 3 hr of blood incubation. 3, 3 hr of blood incubation with SAC. 4,3 hr of blood incubation with AcD. 5, 3 hr of blood incubation with SAC and Chx. (B) Lane 1, Fresh blood, 0 hr. 2, 3 hr of blood incubation. 3,3 hr of blood incubation with SAC. 4,3 hr of blood incubation with SAC and Chx. 5, 3 hr of blood incubation with Chx.
Vol. 2, No. 6 (November 1990: 464-469)
of TNF-ol induced by SAC was abrogated (less than 4 RU/mL in the experiments mentioned above). DISCUSSION The present study was undertaken because of the data indicating that induction of TNF-(U synthesis in human monocyte culture and in whole blood exposed to lipopolysaccharide (LPS) in vitro6,7 cannot be prevented by AcD treatment. It was suggested that TNF-cr mRNA can be presynthesized in human blood monocytes.6,7 Another explanation for these results was the possibility of storage of presynthesized pro-TNF-a within circulating monocytes. It was previously demonstrated that human blood monocytes isolated from the human blood mononuclear cell fraction by adherence to plastic contained no TNF-a! mRNA detectable by conventional Northern analysis.” However, it could not be excluded that dormant mRNA containing AU rich sequences in the 3’-untranslated region could be very unstable in the absence of appropriate cell activation signals1~16-‘8and could be degraded during monocyte isolation by plastic adherence from blood mononuclear cell fractions. Using continuous Percoll gradient centrifugation and cold separation media for purification of human monocytes, we detected no TNF-a mRNA in RNA samples from freshly isolated monocytes. The same result was obtained when AcD and Chx were added to the blood samples immediately after venepuncture. In addition, presynthesized TNF-cu protein was not detected by ELISA in lysates of freshly isolated monocytes using both monoclonal and polyclonal antibodies, indicating that TNF-(r protein was also not stored in circulating human monocytes. To exclude any possibility of hypothetical dormant TNF-cr mRNA destruction during the monocyte separation procedure we have established a simple technique of RNA isolation from whole blood. The whole blood samples were immediately lysed after venepuncture by the buffer inactivating RNAse activity. Using this approach we detected no TNF-cu mRNA in RNA samples isolated from fresh donor blood. Efficiency of this technique for RNA isolation was proved by the demonstration of TNF-(U mRNA in RNA samples isolated from the whole blood exposed to SAC for 3 hr. Thus, the results of the present study show that circulating human blood monocytes contain no stored pool of TNF-ol mRNA or protein and imply that induction of TNF-ol synthesis in blood monocytes requires TNF-a! gene transcription activation and translation. It was demonstrated that the whole blood as well as Percoll-isolated monocytes incubated alone for 3 hr can produce low but detectable amounts of TNF-a mRNA. “Spontaneous” TNF-a! mRNA accumulation was in-
TNF-a inductionin human monocytes/ 467
creased many-fold in the presence of Chx. It was unforeseen to find that the presence of Chx during monocyte isolation and in the monocyte culture activated by SAC caused no “superinduction” of TNF-a mRNA or caused lowering of TNF-a mRNA level. The data suggest that early pretreatment of monocytes with Chx might interfere with TNF-cr mRNA induction in purified monocyte culture under the influence of the potent activating signal provided by SAC. These data imply that different mechanisms can operate in initiation of TNF-a gene transcription in resting, cultured, and activated monocytes. These results seem to conflict with the previous studies that demonstrated “superinduction” of TNF-a mRNA accumulation by Chx in human monocytes isolated by long-time plastic adherence (i.e., macrophages)15 and in mouse peritoneal macrophages elicited by thioglycolate injection.ig This discrepancy might be due to the use of non-activated blood monocytes freshly isolated by Percoll centrifugation in our experiments. In contrast, addition of Chx to the whole blood samples activated by SAC caused enhancement of TNF-a mRNA accumulation. It can be suggested that differential action of Chx on TNF-a mRNA accumulation in Percoll-isolated monocytes and in the whole blood exposed to SAC might be due to the presence of other leukocytes in the whole blood, which also could accumulate TNF-(U mRNA. The synthesis of TNF-a mRNA by human polymorphonuclear leukocytes,” T cells,21 and NK cells2’ has been described. In the present experiments it was shown that the protocol of RNA synthesis inhibition by AcD used in
previous reports6v7 was insufficient to block TNF-a mRNA induction. According to our preliminary data, it was due to the relative resistance of monocyte RNA
synthesis to the action of AcD. It seems that the absence of TNF-a mRNA in the samples of whole blood demonstrates that there are no
pools of TNF-(U mRNA sufficient for detectable production of TNF-a stored in circulating human blood monocytes. It can be hypothesized that TNF-a mRNA
water at 1 mg/mL and stored at -2OOC. Cycloheximide (Chx) (Calbiochem, La Jolla, CA) was dissolved in sterile water at 1 mg/mL and stored at -20°C. RPMI-1640 was purchased from Gibco. Neutralizing monoclonal antibodies
(MAb) (IgGl) against human TNF-o( were produced by 3C7N hybridoma cells grown in ascites.’ Percoll was from Pharmacia, Uppsala. Continuous Percoll gradient separation was performed as described previously.” A sterile solution of heparin for injection (Richter, Budapest) was used for blood stabilization at 25 IU/mL.
Isolation of Monocytes Mononuclear leukocyteswere isolated from fresh heparinized blood of healthy volunteers using a standard Ficoll/
Hypaque (F/H) protocol at +4”C. In some experiments the portion of the donor blood was collected from the venepuncture system directly into bottles that contained the amount of AcD sufficient to achievea final concentration of 10 pg/mL and was further separated by F/H. Cooled mononuclear leukocytes from F/H were fractionated on a continuous Percoll gradient at +4°C.6,io Isolated monocytes were more than 80% naphtyl acetate esterase-positive (NAE+) (NAE kit from Sigma).
Induction of TNF-cwin Monocyte Cultures Monocytes were washed in cold medium, resuspended in 5% FCS-RPM1 at 1.5 x lo6 to 2.0 x lo6 cells/ml and incubated in 50- or loo-mm polystyrene culture dishes (Flow Laboratories). To induce TNF-or synthesis,sterile SAC suspension was added to the monocyte cultures at a final concentration of 0.001% (v/v). Supernatants were decanted and tested for TNF-(r activity. Cells were harvested with a “rubber policeman” and lysed (seebelow). To inhibit protein synthesis, Chx was added to monocyte cultures at a concentration of 10 pg/mL. To inhibit protein or mRNA synthesis very early before cell isolation procedure, portions of blood of the same donor were collected directly into the tubes that contained Chx (10 pg/mL) or AcD (10 pg/mL). Mononuclear cells were further isolated as described above. When monocytes were isolated from Chx-treated blood, all solutions contained the indicated concentration of the drug.
Whole Blood Incubation in Vitro
analysis in human diseases.
Donor blood was collected directly into 50-mL polypropylene tubes (Flow Laboratories) that contained SAC (for a final concentration of 0.001% v/v), AcD (10 pg/mL), or Chx (10 pg/mL). The tubes were incubated at 37°C in a water bath for 3 hr. A portion of incubated blood was immediately used for RNA extraction, the other portion was sedimented, and the plasma was utilized for detection of TNF-cu.
MATERIALS
TNF-a Bioassay
synthesis may be an attribute of macrophage differentiation of monocytes and is incompatible with the capacity of blood monocytes to circulate. The technique of
TNF-a mRNA isolation from the whole blood samples described here might be useful for TNF-a mRNA
AND
METHODS
Reagents Staphylococcus aureus Cowan 1 (SAC) was prepared as described* at a concentration of 5% in sterile saline and stored at +4”C. Actinomycin D (AcD) (Serva, Heidelberg) was dissolved in ethanol at 1 mg/mL and 5 mg/mL or in sterile
Cytotoxic activity of TNF-(r was measured by conventional L-929 cell cytotoxicity photometric assay in the presence of 1 pg/mL of AcD.” A unit of cytotoxin was defined as the reciprocal of the dilution of cytotoxin preparation causing 50% cytotoxicity and was expressed as RU according to titration against a TNF-o bioactivity standard (25 pg of
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human TNF-(U = 1 RU) provided by the National Institute for Biological Standards and Control, UK. For the neutralization assay,serial dilutions of test sampleswere incubated with an excessof neutralizing monoclonal antibodies (MAb) against human TNF-a! for 1 hr at 37°C before the cytotoxicity assay.
on TNF-a mRNA accumulation in human leukocytes and Dr. Sergei Nedospasov for kindly providing genomic DNA of human TNF-a, which was used for the construction of the TNF-a probe in M 13mpl9 phage.
ELISA Analysis
REFERENCES
Collected cells were lysed in ice-cold phosphate buffer containing 1% NP-40 and protease inhibitors: 1 mM phenylmethane-sulfonylfluoride (PMSF), 1 mM N-ethylmaleimide (NEM), 1 mM diethylenetriamine pentaacetic acid (DTPA). After 3 x 10 set of ultrasonic treatment lysates were centrifuged and supernatants were tested. Monoclonal anti-TNF-cu (3C7N at a concentration of 0.01 mg/mL of sodiumcarbonate buffer, pH 9.5) was added to a 96-well microtest plate and incubated overnight at 4°C. The plates were washed 5 times with PBS-O.05% Tween-20 (PBS-T), and TNF-a! sampleswere added and incubated for 40 min (shaking). After five washings with PBS-T, rabbit IgG against human recombinant TNF-a was added for 40 min. Then the plates were washed 5 times with PBS-T and peroxidase-conjugated goat anti-rabbit Ig in PBS-T with 10% sheep serum and 0.1 mg/mL of mouse Ig was added and the incubation was continued for 40 min. After washing with PBS-T, 0.1 mL of 0.6 mg/mL o-phenylendiamine, HCl and 1 mM H,O, in 0.02 M sodium citrate buffer were added and incubated for 30 min. After addition of 50% H,SO,, the optical density of the samples was measured at 492 nm. The concentration of TNF-(U was calculated against a recombinant human TNF-(U ELISA titration.
Northern Blot Analysis RNA from isolated monocytes was extracted by a conventional procedure.‘* To extract RNA from unfractionated blood, the samples of blood (5 mL) were mixed with an equal volume of hot (60°C) lysis solution containing 8 M guanidine thiocyanate, 50 mM sodium citrate, 1% sarcosyl, and 200 mM 2-mercaptoethanol. To minimize the influence of in vitro manipulations, blood samples representing intact (zero time) circulating monocytes were collected from the venepuncture system directly into tubes containing equal volumes of hot lysis solution. Starting at this point isolation of RNA was done as described in reference 12. Fifteen to twenty-five micrograms of total RNA was subjected to formaldehyde-MOPS denaturing electrophoresis (as described in reference 13). Sample loading and RNA integrity were controlled by ethidium bromide fluorescencephotography of gels (data not shown). As a probe for TNF-(U mRNA we used a TNF-ol gene fragment (positions 5793 to 5928 according to reference 14) inserted in M13mp19 phage DNA and labeled by the primer extension method to the specific activity of lo9 cpm/Fg. All manipulations of RNA, blots, and DNA probes were done essentially as described in reference 13. Autoradiographs were developed after overnight exposure with an intensifying screen. Acknowledgments We would like to thank Dr. Daniela Mannel for kindly providing data concerning the influence of SAC
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