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IMMUNOLOGICAL INVESTIGATIONS, 1 9 ( 5 & 6 ) , 453-461 ( 1 9 9 0 )
REGULATION OF TUMOR NECROSIS FACTOR SECRETION I N LEUKOCYTES FROM ALPHA-1-ANTITRYPSIN DEFICIENT HUMANS P h i l i p Scudert', Paul R. Finlyy2, Brian Y. Shon3, John N. Udall , Denise J. Roe , Anita S-F Chong5 'Arizona Cancer C nter, 2Department o f Pathology, 3 D i v i s i o n o f Respiratory Sciences and the Department o f Pediatrics, Uni e r s i t y o f Arizona and the VA Medical Center, Tucson, Arizona 85724, and t h e Departments o f General Surgery and Immunology, Rush Presbyterian S t . Luke's Medical Center, Chicago, I1 1ino1s.
5
I
ABSTRACT
Alpha-1-antitrypsin (AT) i s one o f several alpha-globulins which have been shown t o be i n h i b i t o r s of human peripheral blood monocyte TNF secretion i n v i t r o . AT deficiency states e x i s t , w i t h i n which i n d i v i d u a l s o f e i t h e r the P i S S o r P i Z Z phenotype have reduced hepatocyte and mononuclear phagocyte AT secretion when compared t o normal PiMM subjects. Here we have compared the capacity o f peripheral blood monocytes o f a l l three phenotypes t o respond t o both enhancers and i n h i b i t o r s o f TNF secretion. A1 1 monocytes exposed t o 1ipopolysaccharide (LPS) , interferon-). (IFN-7) and endotoxin, PGE2, transforming growth factor-p1 , whole plasma alpha-globulins, p u r i f i e d AT and IL-6 responded equally w i t h respect t o the secretion o f TNF. Our f i n d i n g s show t h a t the r e g u l a t i o n o f TNF secretion i n leukocytes from AT d e f i c i e n t humans i s normal and suggest t h a t defective AT secretion alone does not r e s u l t i n the aberrant r e g u l a t i o n o f TNF secretion. Alpha-1-antitrypsin (AT) i s an abundant plasma alpha-globulin and a potent i n h i b i t o r of human leukocyte tumor necrosis f a c t o r (TNF) secretion (1). TNF can be detected on the surfaces o f mononuclear leukocytes (2-4) and may be present as a 233 amino acid pro-TNF molecule which i s anchored i n t o the plasma membrane a t i t s amino terminus (5). Suppression o f TNF secretion by AT may be due t o the i n h i b i t i o n o f one o r more serine
proteases which hydrolyse t h e bond between t h e secreted 157 amino a c i d form and the 76 amino a c i d membrane anchoring domain (6). Suppression o f human leukocyte TNF secretion by serum f a c t o r s which share many physical and chemical p r o p e r t i e s w i t h the plasma alpha-globulins (7) 453 Copyright 0 1990 by Marcel Dekker, Inc.
,
suggests t h a t i n
Immunol Invest Downloaded from informahealthcare.com by Flinders University of South Australia on 01/05/15 For personal use only.
454
SCUDERI ET AL.
the systemic circulation monocyte secretion of this cytokine may be continuously down regulated. Within localized microenvironments of the lymphoid organs the production of alpha-globulins by mononuclear phagocytes (8-10) and the secretion of transforming growth factor j3 (TGFj3) (11) and prostaglandin E2 (PGE2) (12) may collectively play an important role in TNF homeostas i s Human AT i s encoded by a pair of codominant alleles located on chromosome 14 (13-15). The gene product i s a 52 kD glycoprotein which can inhibit a broad range of proteases including both neutral proteases such as neutrophil elastase and cathepsin-G and the metalloenzyme collagenase (16). More than thirty forms of AT have been identified based on isoelectric focusing, these have been categorized using the Pi system (protease inhibitor) (17,18). There are two AT phenotypes, PiSS (19) and PiZZ (20), in which secretion i s reduced. PiZZ individuals have one-sixth the normal plasma AT level and when stimulated with lipopolysaccharide peripheral blood monocytes and alveolar macrophages of this phenotype secrete ten-fold less AT compared to cells from normal PiMM individuals (21). Monocytes from AT deficient individuals provide a means o f testing the contribution of this protease ihibitor in the autoregulation of tumor necrosis factor secretion. Here we have compared the regulation of TNF secretion in monocytes from PiZZ and PiSS humans with cells from normal PiMM individuals. Our findings indicate that the regulation of TNF secretion in leukocytes of each phenotype i s equivalent.
.
MATERIALS AND METHODS Cells and culture conditions: Peripheral blood was collected from all subjects, after informed consent had geen given, by venous puncture using heparanized vaccutainer tubes. Blood was collected from four healthy adult volunteers between the ages of 26 and 38 who were phenotyped as PiMM, and from three PiZZ subjects ages 39-51, and one 71-year-old PiSS individual. AT phenotyping was carried out by isoelectric focusing plasma from leukocyte donors (Smith Kline Bio-Science Laboratories, Phoenix, AZ). Leukocytes were separated on Ficoll-hypaque (Pharmacia, Inc. , Piscataway, NJ) and suspended in complete tissue culture medium consisting of RPMI-1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin and streptomycin. The endotoxin content of complete medium was determine to be 150 p g h l using a Limulus Amoebocyte Lysate assay (Associates of Cape Cod, Woods Hole, MA).
Immunol Invest Downloaded from informahealthcare.com by Flinders University of South Australia on 01/05/15 For personal use only.
REGULATION OF TUMOR NECROSIS FACTOR SECRETION
3.0
0-0
PiSS
2.5
0-0
PiMM
&I
B-• A-A
k E c
455
$1
2.0 1.5
LL
f
1 .o
0.5 0.0
I
3.01 2.5
0.0
1
10
100
1000
IFN-7 U/rnl
Figure 1 Figure 1. Stimulation of human peripheral blood leukocyte TNF secretion with either LPS or IFN-7. Ficoll s parated peripheral blood mononuclear leukocytes were suspended at 1 x 105/ml in complete medium and the indicated concentration of either LPS or recombinant human IFN-7 was added. After eighteen hours the culture supernatants were harvested and assayed for secreted TNF by ELISA. One representative experiment is shown and the values displayed are the mean of three replicates t standard deviation.
SCUDERI ET AL.
456
0-0
PiZZ
A-A Immunol Invest Downloaded from informahealthcare.com by Flinders University of South Australia on 01/05/15 For personal use only.
1 .oo 1
0-0 0-0
PiSS
0-0 A-A
PiMM
B-¤
J
0.00 0.0
1 2.5
5.0
10.0
TGFBl ng/rnl
Figure 2 Figure 2. Suppression of leukocyte TNF secretion. Ficoll separated peripheral blood ononuclear cells were suspended in complete medium at a density of 1 x 10'k/ml and exposed to the indicated concentration of inhibitor for eighteen hours. Culture supernatants were assayed for secreted TNF by ELISA. The values displayed represent the mean of three replicates 2 standard deviation.
457
Immunol Invest Downloaded from informahealthcare.com by Flinders University of South Australia on 01/05/15 For personal use only.
REGULATION OF TUMOR NECROSIS FACTOR SECRETION
Y
0.004 0.0
0.31
0.63
1.25
ALPHA-GLOBULINS mg/ml
0.751
ALPHA- 1 -ANTITRYPSIN mg/ml
Figure 2 continued
Reagents: Whole plasma alpha-globulins and purified AT were purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant human interferon-7 (IFN-7), TNF and monoclonal antibody 6E which is specific for TNF were gifts o f Genentech, Inc. (S. San Francisco, CA). Recombinant IL-6 was a gift of Genetics Institute (Boston, MA). Polyclonal antiserum specific for human TNF was produced in New Zealand white rabbits as previously described (22). Rabbit antisera specific for either human AT or alpha-2macroglobul in (MC) were purchased from Sigma. Lipopolysaccharide (LPS) extracted from E. coli Olll:B4 was purchased from Difco Inc. (Detroit, MI).
Immunol Invest Downloaded from informahealthcare.com by Flinders University of South Australia on 01/05/15 For personal use only.
458
SCUDERI ET AL.
Procine transforming growth factor pl (TGF pl) was purchased from R&D Systems Inc. (Minneapolis, MN). TNF ELISA: An ELISA specific for human TNF was used to quantitate this cytokine In supernatants from cultures containing 1 x lo5 Ficollseparated leukocytes which had been incubated in 200 pl of fluid for 18 hours. The details of this assay have been published (23). The lower limit of TNF detection with this assay system is 40 pg/ml. RESULTS AND DISCUSSION Stimulation of leukocyte TNF secretion: Our previous study demonstrated that exogenous alpha-globul ins added to cultured 1 eukocytes blocked TNF secretion and that AT was the most effective alpha-globul in tested (1). The AT deficiency in the PiZZ phenotype is due to a defect in AT secretion rather than production (27). An amino acid substitution of lysine for glutamic acid at position 342 in the AT molecule is believed to cause the newly translated protein to fold improperly in the endoplastic reticulum reducing AT migration to the golgi and ultimately resulting in reduced extracellular transport (28,29). Leukocytes of the PiZZ phenotype offer a unique opportunity to test the role of AT in the autoregulation of TNF secretion. Human peripheral blood monocyte TNF secretion can normally be stimulated by either bacterial endotoxin (24,25) or IFN-7 in combination with endotoxin (26). If endogenous AT plays a significant role in TNF autoregulation one would expect that PiZZ leukocytes would respond to inducers of cytokine secretion with increased TNF release. Figure 1 shows that when Fi col 1 -hypaque separated 1 eukocytes from AT deficient Pi ZZ and PiSS donors stimulated with either LPS or IFN-7 secreted quantities of TNF comparable to that produced by leukocytes from normal PiMM individuals. The fact that we did not see enhanced secretion of TNF in leukocytes o f the PiZZ and PiSS phenotype may be a reflection of the combined regulatory activity of alpha-globulinsl other than AT, and additional elements such as TGF-p and PGE2. Although the PiZZ monocytes secrete reduced quantities of AT, the production and secretion of MC is unaltered in these cells (30). The combined effect of MC and other monocyte derived regulators may ultimately be more important in TNF homeostasis than any individual agent such as AT. Suppression of leukocyte TNF secretion: TNF secretion can normally be i nhi bi ted by adding prostaglandin E2 (12) transforming growth factor p (ll), and several plasma alpha-globulins (1) to leukocytes in vitro.
459
REGULATION O F TUMOR NECROSIS FACTOR SECRETION
0-0 A-A
PiZZ
Immunol Invest Downloaded from informahealthcare.com by Flinders University of South Australia on 01/05/15 For personal use only.
0-0
IL-6
CONCENTRATION
ng/ml
Figure 3 Figure 3. Effect of IL-6 on leukocyte TNF secretion. Ficoll separated peripheral blood mononucl ear cell s were suspended in compl ete medi um containing the indicated amount of recombinant human IL-6, at a density of 1 x lo6 cells/ml. After four hours at 37OC LPS 0111:84 was added to a final concentration of 1 p g h l and the cells were incubated for an additional 12 hours. Culture supernatants were assayed for secreted TNF by ELISA. The values displayed represent the mean of four replicates 2 standard deviation.
Figure 2 shows that leukocytes of all phenotypes were equally suppressible by PGE2, TGFp1, whole alpha-globulins and purified AT. IL-6 has recently been reported to suppress human monocyte TNF secretion in a concentration dependent manner (31,32). The addition of IL-6 to both PiZZ and PiMM leukocyte suppressed TNF release equally (Figure 3). Thus the leukocytes of both phenotype are equally responsive to exogenous inhibitors. Our findings show that a single defect in endogenous inhibitor production such as AT does not result in increased and uncontrolled TNF secretion when monocytes are stimulated. The variety of monocyte derived suppressive factors now known to reduce or completely inhibit TNF secretion certainly argues in favor of a complex system of overlapping regulatory elements. Further, the involvement of TNF in inflammatory processes (3336) and the potentially lethal consequences of large scale and unregulated activation of TNF secretion almost certainly necessitates such regulatory complexity in cells of the reticuloendothelial system.
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REFERENCES P. Scuderi, R.T.
2.
Raitano and J. Rybski, Eur. J. Immunol., l9, 939-942 (1989). T. Decker, M.L. Lohmann-Matthes and G.E. G i f f o r d , J. Immunol.,
3. 4. 5.
Dorr, J.D.
L i d d i l , P.R.
Finley, P. Meltzer, A.B.
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957-962 (1987). R.S. Kornbluth and T.S. Edington, J. Immunol 137, 2585-2591 (1986). J.L. Urban, H.M. Shepard, J.L. Rothstein, B.J. Sugarman and H. Schreiber, Proc. Natl. Acad. Sci. USA, 83, 5233-5237 (1986). M. Kriegler, C. Perez, K. DeFay, I. Albert and S.O. Lu, C e l l , 53, 45-
.,
53 (1988). 6. 7.
J. Immunol., 143, 168-173 (1989). P. Scuderi, R.A. Rippe, A.B. Raitano and J. Rybski, J. Leukocyte
P. Scuderi, BiOl
8. 9. 10.
34-40 (1989).
P. Isaacson, D.B.
Jones, G.H.
Millward-Sadler, M.A.
J. Clin. Path., 34, 982-990 (1981). R. Van Furth, J.A. Kramps and M.M.C.
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(1977). Judd and S. Payne,
Diesselhof-Den Dulk, Clin. Exp.
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Immunol , 5 l , 551-557 (1983). T. Espevik, I.S. F i g a r i , M.R. Shalaby, G.A. Lackides, G.D. Lewis, H.M. Shepard and M.A. Palladino, J. Exp. Med., 166, 571-576 (1987). S.L. Kunkel, R.C. Wiggins, S.W. Chensue and J. Larrick, Biochem.
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Biophys. Res. Commun., 137, 404-410 (1986). G.J. Darlington, K.H. Astrin, S.P. Muirhead, R.J.
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Smith, Proc. Natl. Acad. Sci. USA, 79, 870-873 (1982). V.J. Kidd, R.B. Wallace, K. I t a k u r a and S.L. Woo, Nature,
11.
15. 16.
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T. Hovi, D. Mosher and A. Vaheri, J. Exp. Med.,
17. 18. 19. 20.
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Desnick and M.
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(1983) M. Leicht, G.L. Long, T. Chandra, K. Kurachi, V.J. Kidd, M. Mace, E.W. Davie and S.L. Woo, Nature, E l 655-659 (1982) A. Koj, i n The acute-phase response t o i n j u r y and i n f e c t i o n - Research monographs i n c e l l and t i s s u e physiology, Vol. 10, A.H. Gordon and A. Koj, eds. Elsevier Science Publishing Co., Amsterdam (1981) pp. 145160. D.W. Cox, A.M. Johnson and M.K. Fagerhol, Hum. Genet., 3, 429-433 (1980) M.K. Fagerhol and D.W. Cox, Adv. Hum. Genet., ll, 1-62 (1981). F. Ogushi, R.C. Hubbard, G.A. F e l l s , A. Casolaro, D.T. Curiel, M.L. Brantly and R.G. Crystal, Am. Rev. Respir. D i s . , 137, 364-370 (1988). F. Kueppers and L.F. Black, Am. Rev. Respir. Dis., 110, 176-194
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(1974)
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21. J.F.
Mornex, A. Chytil-Weir, Y. Martinet, M. Courtney, J.P.
Crystal, J. Clin. Invest., P. Scuderi, K.E. S t e r l i n g , A.B.
R.G.
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22.
77, 1952-1961 (1986). Raitano, T.M. Grogan and R.A.
LeCocq and Rippe,
J. I n t e r f e r o n Res., I-, 155-164 (1987). 23. P. Scuderi, K.E. S t e r l i n g , K.S. Lam, P.R. Finley, K.J. Ryan, C.G. Ray, E. Peterson, D.J. Slymen and S.E. Salmon, Lancet, ii, 1364-1365, 1986. 24. E.A. Carswell, L.J. Old, R.L. Kassel, S. Green, N. F i o r e and B. Williamson, Proc. Natl. Acad. Sci. USA, 72, 3666-3670 (1975). 25. G.E. G i f f o r d and M.L. Lohmann-Matthes, I n t l . J. Cancer, 38, 135-137
.
(1986)
26. R. P h i l i p and L.B. Epstein, Nature, 323, 86-89 (1986). 27. J. Lieberman, i n I n h e r i t e d diseases o f the lung: A l p h a l - a n t i t r y p s i n deficiency, W.B. Saunders Co., Philadelphia, (1988) pp 99-133. 28. R.W. C a r r e l l , J.O. Jeppsson, C.B. Laurell, S.O. Brennan, M.C. Owen, L. Vaughn and D.R. Boswell, Nature, 298, 329-334 (1982). 29. H. Loebermann, R. Tokuoka, J. Deisenhofer and R. Huber, J. Mol. Biol., 177, 531-557 (1984). 30. J.E. Gadek, G.A. F e l l s , R.L. Zimmerman and S.I. Rennard, J. Clin. Invest. , 68, 889-898 (1981). 31. D. Aderka, J. Le, J. Vilcek, J. Immunol., 143, 3517-3523 (1989). 32. R. Schindler, J. Mancilla, S. Enders, R. Ghorbani, S.C. Clark, C.A. D i narel l o , B1ood , 75: 40-47 (1990). 33. B. Beutler, I.W. Milsark and A. Cerami, Science, 229, 869-871 (1985). 34. G.J. Darlington, D.R. Wilson and L.B. Lachman, J. Cell. Biol., 103, 787-793 (1986). 35. C.A. Dlnarello, J.G. Cannon, S.M. Wolff, H.A. Bernheim, B. Beutler, A. Cerami, I.S.
F i g a r i , M.A.
Palladino and J.V.
O'Connor, J. Exp. Med.,
163, 1433-1450 (1986). 36. K.J. Tracey, B. Beutler, S.F.
Lowry, J. Merryweather, S. Wolpe, I.W. Mllsark, R.J. H a r i r i , T.J. Fahey, A. Zentella, J.D. Albert, G.T. Shires and A. Cerami , Science, 234, 470-474 (1986).
IMMUNOLOGICAL INVESTIGATIONS, 1 9 ( 5 & 6 ) , 453-461 ( 1 9 9 0 )
REGULATION OF TUMOR NECROSIS FACTOR SECRETION I N LEUKOCYTES FROM ALPHA-1-ANTITRYPSIN DEFICIENT HUMANS P h i l i p Scudert', Paul R. Finlyy2, Brian Y. Shon3, John N. Udall , Denise J. Roe , Anita S-F Chong5 'Arizona Cancer C nter, 2Department o f Pathology, 3 D i v i s i o n o f Respiratory Sciences and the Department o f Pediatrics, Uni e r s i t y o f Arizona and the VA Medical Center, Tucson, Arizona 85724, and t h e Departments o f General Surgery and Immunology, Rush Presbyterian S t . Luke's Medical Center, Chicago, I1 1ino1s.
5
I
ABSTRACT
Alpha-1-antitrypsin (AT) i s one o f several alpha-globulins which have been shown t o be i n h i b i t o r s of human peripheral blood monocyte TNF secretion i n v i t r o . AT deficiency states e x i s t , w i t h i n which i n d i v i d u a l s o f e i t h e r the P i S S o r P i Z Z phenotype have reduced hepatocyte and mononuclear phagocyte AT secretion when compared t o normal PiMM subjects. Here we have compared the capacity o f peripheral blood monocytes o f a l l three phenotypes t o respond t o both enhancers and i n h i b i t o r s o f TNF secretion. A1 1 monocytes exposed t o 1ipopolysaccharide (LPS) , interferon-). (IFN-7) and endotoxin, PGE2, transforming growth factor-p1 , whole plasma alpha-globulins, p u r i f i e d AT and IL-6 responded equally w i t h respect t o the secretion o f TNF. Our f i n d i n g s show t h a t the r e g u l a t i o n o f TNF secretion i n leukocytes from AT d e f i c i e n t humans i s normal and suggest t h a t defective AT secretion alone does not r e s u l t i n the aberrant r e g u l a t i o n o f TNF secretion. Alpha-1-antitrypsin (AT) i s an abundant plasma alpha-globulin and a potent i n h i b i t o r of human leukocyte tumor necrosis f a c t o r (TNF) secretion (1). TNF can be detected on the surfaces o f mononuclear leukocytes (2-4) and may be present as a 233 amino acid pro-TNF molecule which i s anchored i n t o the plasma membrane a t i t s amino terminus (5). Suppression o f TNF secretion by AT may be due t o the i n h i b i t i o n o f one o r more serine
proteases which hydrolyse t h e bond between t h e secreted 157 amino a c i d form and the 76 amino a c i d membrane anchoring domain (6). Suppression o f human leukocyte TNF secretion by serum f a c t o r s which share many physical and chemical p r o p e r t i e s w i t h the plasma alpha-globulins (7) 453 Copyright 0 1990 by Marcel Dekker, Inc.
,
suggests t h a t i n
Immunol Invest Downloaded from informahealthcare.com by Flinders University of South Australia on 01/05/15 For personal use only.
454
SCUDERI ET AL.
the systemic circulation monocyte secretion of this cytokine may be continuously down regulated. Within localized microenvironments of the lymphoid organs the production of alpha-globulins by mononuclear phagocytes (8-10) and the secretion of transforming growth factor j3 (TGFj3) (11) and prostaglandin E2 (PGE2) (12) may collectively play an important role in TNF homeostas i s Human AT i s encoded by a pair of codominant alleles located on chromosome 14 (13-15). The gene product i s a 52 kD glycoprotein which can inhibit a broad range of proteases including both neutral proteases such as neutrophil elastase and cathepsin-G and the metalloenzyme collagenase (16). More than thirty forms of AT have been identified based on isoelectric focusing, these have been categorized using the Pi system (protease inhibitor) (17,18). There are two AT phenotypes, PiSS (19) and PiZZ (20), in which secretion i s reduced. PiZZ individuals have one-sixth the normal plasma AT level and when stimulated with lipopolysaccharide peripheral blood monocytes and alveolar macrophages of this phenotype secrete ten-fold less AT compared to cells from normal PiMM individuals (21). Monocytes from AT deficient individuals provide a means o f testing the contribution of this protease ihibitor in the autoregulation of tumor necrosis factor secretion. Here we have compared the regulation of TNF secretion in monocytes from PiZZ and PiSS humans with cells from normal PiMM individuals. Our findings indicate that the regulation of TNF secretion in leukocytes of each phenotype i s equivalent.
.
MATERIALS AND METHODS Cells and culture conditions: Peripheral blood was collected from all subjects, after informed consent had geen given, by venous puncture using heparanized vaccutainer tubes. Blood was collected from four healthy adult volunteers between the ages of 26 and 38 who were phenotyped as PiMM, and from three PiZZ subjects ages 39-51, and one 71-year-old PiSS individual. AT phenotyping was carried out by isoelectric focusing plasma from leukocyte donors (Smith Kline Bio-Science Laboratories, Phoenix, AZ). Leukocytes were separated on Ficoll-hypaque (Pharmacia, Inc. , Piscataway, NJ) and suspended in complete tissue culture medium consisting of RPMI-1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin and streptomycin. The endotoxin content of complete medium was determine to be 150 p g h l using a Limulus Amoebocyte Lysate assay (Associates of Cape Cod, Woods Hole, MA).
Immunol Invest Downloaded from informahealthcare.com by Flinders University of South Australia on 01/05/15 For personal use only.
REGULATION OF TUMOR NECROSIS FACTOR SECRETION
3.0
0-0
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2.0 1.5
LL
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1 .o
0.5 0.0
I
3.01 2.5
0.0
1
10
100
1000
IFN-7 U/rnl
Figure 1 Figure 1. Stimulation of human peripheral blood leukocyte TNF secretion with either LPS or IFN-7. Ficoll s parated peripheral blood mononuclear leukocytes were suspended at 1 x 105/ml in complete medium and the indicated concentration of either LPS or recombinant human IFN-7 was added. After eighteen hours the culture supernatants were harvested and assayed for secreted TNF by ELISA. One representative experiment is shown and the values displayed are the mean of three replicates t standard deviation.
SCUDERI ET AL.
456
0-0
PiZZ
A-A Immunol Invest Downloaded from informahealthcare.com by Flinders University of South Australia on 01/05/15 For personal use only.
1 .oo 1
0-0 0-0
PiSS
0-0 A-A
PiMM
B-¤
J
0.00 0.0
1 2.5
5.0
10.0
TGFBl ng/rnl
Figure 2 Figure 2. Suppression of leukocyte TNF secretion. Ficoll separated peripheral blood ononuclear cells were suspended in complete medium at a density of 1 x 10'k/ml and exposed to the indicated concentration of inhibitor for eighteen hours. Culture supernatants were assayed for secreted TNF by ELISA. The values displayed represent the mean of three replicates 2 standard deviation.
457
Immunol Invest Downloaded from informahealthcare.com by Flinders University of South Australia on 01/05/15 For personal use only.
REGULATION OF TUMOR NECROSIS FACTOR SECRETION
Y
0.004 0.0
0.31
0.63
1.25
ALPHA-GLOBULINS mg/ml
0.751
ALPHA- 1 -ANTITRYPSIN mg/ml
Figure 2 continued
Reagents: Whole plasma alpha-globulins and purified AT were purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant human interferon-7 (IFN-7), TNF and monoclonal antibody 6E which is specific for TNF were gifts o f Genentech, Inc. (S. San Francisco, CA). Recombinant IL-6 was a gift of Genetics Institute (Boston, MA). Polyclonal antiserum specific for human TNF was produced in New Zealand white rabbits as previously described (22). Rabbit antisera specific for either human AT or alpha-2macroglobul in (MC) were purchased from Sigma. Lipopolysaccharide (LPS) extracted from E. coli Olll:B4 was purchased from Difco Inc. (Detroit, MI).
Immunol Invest Downloaded from informahealthcare.com by Flinders University of South Australia on 01/05/15 For personal use only.
458
SCUDERI ET AL.
Procine transforming growth factor pl (TGF pl) was purchased from R&D Systems Inc. (Minneapolis, MN). TNF ELISA: An ELISA specific for human TNF was used to quantitate this cytokine In supernatants from cultures containing 1 x lo5 Ficollseparated leukocytes which had been incubated in 200 pl of fluid for 18 hours. The details of this assay have been published (23). The lower limit of TNF detection with this assay system is 40 pg/ml. RESULTS AND DISCUSSION Stimulation of leukocyte TNF secretion: Our previous study demonstrated that exogenous alpha-globul ins added to cultured 1 eukocytes blocked TNF secretion and that AT was the most effective alpha-globul in tested (1). The AT deficiency in the PiZZ phenotype is due to a defect in AT secretion rather than production (27). An amino acid substitution of lysine for glutamic acid at position 342 in the AT molecule is believed to cause the newly translated protein to fold improperly in the endoplastic reticulum reducing AT migration to the golgi and ultimately resulting in reduced extracellular transport (28,29). Leukocytes of the PiZZ phenotype offer a unique opportunity to test the role of AT in the autoregulation of TNF secretion. Human peripheral blood monocyte TNF secretion can normally be stimulated by either bacterial endotoxin (24,25) or IFN-7 in combination with endotoxin (26). If endogenous AT plays a significant role in TNF autoregulation one would expect that PiZZ leukocytes would respond to inducers of cytokine secretion with increased TNF release. Figure 1 shows that when Fi col 1 -hypaque separated 1 eukocytes from AT deficient Pi ZZ and PiSS donors stimulated with either LPS or IFN-7 secreted quantities of TNF comparable to that produced by leukocytes from normal PiMM individuals. The fact that we did not see enhanced secretion of TNF in leukocytes o f the PiZZ and PiSS phenotype may be a reflection of the combined regulatory activity of alpha-globulinsl other than AT, and additional elements such as TGF-p and PGE2. Although the PiZZ monocytes secrete reduced quantities of AT, the production and secretion of MC is unaltered in these cells (30). The combined effect of MC and other monocyte derived regulators may ultimately be more important in TNF homeostasis than any individual agent such as AT. Suppression of leukocyte TNF secretion: TNF secretion can normally be i nhi bi ted by adding prostaglandin E2 (12) transforming growth factor p (ll), and several plasma alpha-globulins (1) to leukocytes in vitro.
459
REGULATION O F TUMOR NECROSIS FACTOR SECRETION
0-0 A-A
PiZZ
Immunol Invest Downloaded from informahealthcare.com by Flinders University of South Australia on 01/05/15 For personal use only.
0-0
IL-6
CONCENTRATION
ng/ml
Figure 3 Figure 3. Effect of IL-6 on leukocyte TNF secretion. Ficoll separated peripheral blood mononucl ear cell s were suspended in compl ete medi um containing the indicated amount of recombinant human IL-6, at a density of 1 x lo6 cells/ml. After four hours at 37OC LPS 0111:84 was added to a final concentration of 1 p g h l and the cells were incubated for an additional 12 hours. Culture supernatants were assayed for secreted TNF by ELISA. The values displayed represent the mean of four replicates 2 standard deviation.
Figure 2 shows that leukocytes of all phenotypes were equally suppressible by PGE2, TGFp1, whole alpha-globulins and purified AT. IL-6 has recently been reported to suppress human monocyte TNF secretion in a concentration dependent manner (31,32). The addition of IL-6 to both PiZZ and PiMM leukocyte suppressed TNF release equally (Figure 3). Thus the leukocytes of both phenotype are equally responsive to exogenous inhibitors. Our findings show that a single defect in endogenous inhibitor production such as AT does not result in increased and uncontrolled TNF secretion when monocytes are stimulated. The variety of monocyte derived suppressive factors now known to reduce or completely inhibit TNF secretion certainly argues in favor of a complex system of overlapping regulatory elements. Further, the involvement of TNF in inflammatory processes (3336) and the potentially lethal consequences of large scale and unregulated activation of TNF secretion almost certainly necessitates such regulatory complexity in cells of the reticuloendothelial system.
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