Murine α-Macroglobulins Demonstrate Divergent Activities as Neutralizers of Transforming Growth Factor-β and as Inducers of Nitric Oxide Synthesis
1996; Elsevier BV; Volume: 271; Issue: 40 Linguagem: Inglês
10.1074/jbc.271.40.24982
ISSN1083-351X
AutoresDonna J. Webb, Janice Wen, Jeffrey J. Lysiak, Lieve Umans, Fred Van Leuven, Steven L. Gonias,
Tópico(s)Cancer, Hypoxia, and Metabolism
Resumoα2-Macroglobulin null mice demonstrate increased resistance to endotoxin challenge (Umans, L., Serneels, L., Overbergh, L., Van Leuven, F., and Van den Berghe, H. (1995) J. Biol. Chem. 270, 19778-19785). We hypothesized that this phenotype might reflect the function of murine α2M (mα2M) as a neutralizer of transforming growth factor-β (TGF-β) and inducer of nitric oxide synthesis in vivo. When incubated with wild-type mouse plasma, TGF-β1 and TGF-β2 bound only to mα2M. Alternative TGF-β-binding proteins were not detected in plasma from α2M(−/−) mice. Wild-type mouse plasma, but not plasma from α2M(−/−) mice, inhibited TGF-β1 binding to TGF-β receptors on fibroblasts. Purified mα2M bound TGF-β1 and TGF-β2 with similar affinity; the KD values were 28 ± 4 and 33 ± 4 nM, respectively. Murinoglobulin, the second murine α-macroglobulin, bound both TGF-β isoforms with 30-fold lower affinity. Mα2M counteracted the activities of TGF-β1 and TGF-β2 in an endothelial cell growth assay. Mα2M also induced NO synthesis when incubated with RAW 264.7 cells, an activity which probably results from the neutralization of autocrine TGF-β activity. Human α2M induced NO synthesis comparably to mα2M; however, MUG had no effect. These studies demonstrate that the ability to neutralize TGF-β is a property of mα2M, which is not redundant in the murine α-macroglobulin family or in murine plasma. Mα2M is the only murine α-macroglobulin that promotes NO synthesis. The absence of mα2M, in α2M(−/−) mice, may allow TGF-β to more efficiently suppress excessive iNOS expression following endotoxin challenge. α2-Macroglobulin null mice demonstrate increased resistance to endotoxin challenge (Umans, L., Serneels, L., Overbergh, L., Van Leuven, F., and Van den Berghe, H. (1995) J. Biol. Chem. 270, 19778-19785). We hypothesized that this phenotype might reflect the function of murine α2M (mα2M) as a neutralizer of transforming growth factor-β (TGF-β) and inducer of nitric oxide synthesis in vivo. When incubated with wild-type mouse plasma, TGF-β1 and TGF-β2 bound only to mα2M. Alternative TGF-β-binding proteins were not detected in plasma from α2M(−/−) mice. Wild-type mouse plasma, but not plasma from α2M(−/−) mice, inhibited TGF-β1 binding to TGF-β receptors on fibroblasts. Purified mα2M bound TGF-β1 and TGF-β2 with similar affinity; the KD values were 28 ± 4 and 33 ± 4 nM, respectively. Murinoglobulin, the second murine α-macroglobulin, bound both TGF-β isoforms with 30-fold lower affinity. Mα2M counteracted the activities of TGF-β1 and TGF-β2 in an endothelial cell growth assay. Mα2M also induced NO synthesis when incubated with RAW 264.7 cells, an activity which probably results from the neutralization of autocrine TGF-β activity. Human α2M induced NO synthesis comparably to mα2M; however, MUG had no effect. These studies demonstrate that the ability to neutralize TGF-β is a property of mα2M, which is not redundant in the murine α-macroglobulin family or in murine plasma. Mα2M is the only murine α-macroglobulin that promotes NO synthesis. The absence of mα2M, in α2M(−/−) mice, may allow TGF-β to more efficiently suppress excessive iNOS expression following endotoxin challenge. INTRODUCTIONBacterial endotoxin is a lipopolysaccharide complex derived from the outer membranes of Gram-negative bacteria and a major mediator of the pathophysiologic changes referred to as septic shock (1Raetz C.R.H. Ulevitch R.J. Wright S.D. Sibley C.H. Ding A. Nathan C.F. FASEB J. 1991; 5: 2652-2660Google Scholar, 2Parrillo J.E. N. Engl. J. Med. 1993; 328: 1471-1477Google Scholar). Endotoxin triggers release of inflammatory cytokines, including tumor necrosis factor-α and interleukin-1β, from a number of cell types, including macrophages and endothelial cells (1Raetz C.R.H. Ulevitch R.J. Wright S.D. Sibley C.H. Ding A. Nathan C.F. FASEB J. 1991; 5: 2652-2660Google Scholar, 3Burrell R. Circ. Shock. 1994; 43: 137-153Google Scholar). These cytokines, in addition to endotoxin, induce increased cellular synthesis of nitric oxide (4Nathan C. Xie Q. J. Biol. Chem. 1994; 269: 13725-13728Google Scholar, 5Wolfe T.A. Dasta J.F. Ann. Pharmacother. 1995; 29: 36-46Google Scholar, 6Kilbourn R.G. Gross S.S. Jubran A. Adams J. Griffith O.W. Levi R. Lodato R.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3629-3632Google Scholar, 7Cunha F.Q. Assreuy J. Moss D.W. Rees D. Leal L.M.C. Moncada S. Carrier M. O'Donnell C.A. Liew F.Y. Immunology. 1994; 81: 211-215Google Scholar). NO 1The abbreviations used are: NOnitric oxideNOSnitric oxide synthaseiNOSinducible nitric oxide synthasehα2Mhuman α2-macroglobulinmα2Mmurine α2-macroglobulinMUGmurinoglobulinTGF-βtransforming growth factor-βLRPlow density lipoprotein receptor-related proteinFBSfetal bovine serumBS3bis(sulfosuccinimidyl) suberateFBHEfetal bovine heart endothelialPAGEpolyacrylamide gel electrophoresis. plays a significant role in mediating the profound vasodilation, hypotension, and hyporeactivity to pressor agents, which are characteristic of severe septic shock (2Parrillo J.E. N. Engl. J. Med. 1993; 328: 1471-1477Google Scholar, 5Wolfe T.A. Dasta J.F. Ann. Pharmacother. 1995; 29: 36-46Google Scholar, 6Kilbourn R.G. Gross S.S. Jubran A. Adams J. Griffith O.W. Levi R. Lodato R.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3629-3632Google Scholar, 7Cunha F.Q. Assreuy J. Moss D.W. Rees D. Leal L.M.C. Moncada S. Carrier M. O'Donnell C.A. Liew F.Y. Immunology. 1994; 81: 211-215Google Scholar, 8Petros A. Lamb G. Leone A. Moncada S. Bennett D. Vallance P. Cardiovasc. Res. 1994; 28: 34-39Google Scholar, 9Szabo C. Mitchell J.A. Thiemermann C. Vane J.R. Br. J. Pharmacol. 1993; 108: 786-792Google Scholar).NO is produced by three enzymes of the nitric oxide synthase (NOS) family (4Nathan C. Xie Q. J. Biol. Chem. 1994; 269: 13725-13728Google Scholar, 10Nathan C. Xie Q.-W. Cell. 1994; 78: 915-918Google Scholar). Two constitutive forms of NOS (NOS-I, NOS-III) produce low levels of NO in response to agonists which increase intracellular calcium. The resulting NO is important in the regulation of normal homeostatic processes, including blood pressure and neuronal transmission (10Nathan C. Xie Q.-W. Cell. 1994; 78: 915-918Google Scholar). The inducible form of NOS (iNOS or NOS-II) produces high levels of NO, independently of intracellular calcium. The NO produced by iNOS functions in the immune response to pathogens and may also provide cytotoxic activity toward cancer cells (10Nathan C. Xie Q.-W. Cell. 1994; 78: 915-918Google Scholar). Since iNOS activity is not calcium-dependent, regulation of iNOS expression is critical and many cytokines are involved in this process in various cell types (11De Vera M.E. Shapiro R.A. Nussler A.K. Mudgett J.S. Simmons R.L. Morris S.M. Billiar T.R. Geller D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1054-1059Google Scholar).Intravenous administration of endotoxin in experimental animals causes an initial, transient increase in NO that is probably due to stimulation of constitutive NOS followed by a sustained increase in NO produced by iNOS (8Petros A. Lamb G. Leone A. Moncada S. Bennett D. Vallance P. Cardiovasc. Res. 1994; 28: 34-39Google Scholar). Mice that are iNOS-deficient, due to gene disruption in embryonic stem cells, demonstrate resistance to doses of endotoxin that are lethal in wild-type animals (12MacMicking J.D. Nathan C. Hom G. Chartrain N. Fletcher D.S. Trumbauer M. Stevens K. Xie Q.-W. Sokol K. Hutchinson N. Chin H. Mudgett J.S. Cell. 1995; 81: 641-650Google Scholar, 13Wei X.Q. Charles I.G. Smith A. Ure J. Feng G.-J. Huang F.P. Xu D. Muller W. Moncada S. Liew F.Y. Nature. 1995; 375: 408-411Google Scholar). iNOS(−/−) mice are also less effective at eliminating infections caused by Listeria monocytogenes or Leishmania major. Macrophages that are isolated from iNOS(−/−) mice are less effective killers of cancer cells in vitro (12MacMicking J.D. Nathan C. Hom G. Chartrain N. Fletcher D.S. Trumbauer M. Stevens K. Xie Q.-W. Sokol K. Hutchinson N. Chin H. Mudgett J.S. Cell. 1995; 81: 641-650Google Scholar).Human α2-macroglobulin (hα2M) is a proteinase inhibitor and specific cytokine carrier that induces iNOS expression and NO synthesis in the murine macrophage-like cell line, RAW 264.7 (14Lysiak J.J. Hussaini I.M. Webb D.J. Glass II, W.F. Allietta M. Gonias S.L. J. Biol. Chem. 1995; 270: 21919-21927Google Scholar). The mechanism of iNOS induction involves the neutralization of autocrine cytokine activity by hα2M. Since RAW 264.7 cells synthesize, secrete, and activate transforming growth factor-β (TGF-β) and because TGF-β-neutralizing antibody induces RAW 264.7 cell NO synthesis, similarly to hα2M, we proposed that neutralization of autocrine TGF-β is most likely responsible for the increase in iNOS expression in hα2M-treated RAW 264.7 cells (14Lysiak J.J. Hussaini I.M. Webb D.J. Glass II, W.F. Allietta M. Gonias S.L. J. Biol. Chem. 1995; 270: 21919-21927Google Scholar).Hα2M exists in at least two well defined conformations (15Barrett A.J. Starkey P.M. Biochem. J. 1973; 133: 709-724Google Scholar, 16Gonias S.L. Reynolds J.A. Pizzo S.V. Biochim. Biophys. Acta. 1982; 705: 306-314Google Scholar). The native form is active as a proteinase inhibitor, but not recognized by the α2M receptor, low density lipoprotein receptor-related protein (LRP) (17Pizzo S.V. Gonias S.L. Conn P.M. The Receptors. Academic Press, Inc., New York1984: 177Google Scholar). Thus, native α2M is stable in the blood and interstitial spaces. The activated form of α2M, generated by reaction with proteinase, is LRP-recognized. In the RAW 264.7 cell system, native α2M and activated α2M are comparably effective as inducers of NO synthesis (14Lysiak J.J. Hussaini I.M. Webb D.J. Glass II, W.F. Allietta M. Gonias S.L. J. Biol. Chem. 1995; 270: 21919-21927Google Scholar).The murine α-macroglobulin family includes at least five distinct genes, two of which encode for proteins that have been purified and characterized (18Overbergh L. Torrekens S. Van Leuven F. Van den Berghe H. J. Biol. Chem. 1991; 266: 16903-16910Google Scholar, 19Van Leuven F. Torrekens S. Overbergh L. Lorent K. De Strooper B. Van den Berghe H. Eur. J. Biochem. 1992; 210: 319-327Google Scholar, 20Overbergh L. Hilliker C. Lorent K. Van Leuven F. Van den Berghe F. Genomics. 1994; 22: 530-539Google Scholar). Murine α2M (mα2M) is a tetrameric protein (Mr ~ 720,000) and a close homologue of human α2M. Hα2M and mα2M share comparably broad proteinase-inhibitory specificities and undergo equivalent proteinase-induced conformational changes. The second member of the murine α-macroglobulin family is murinoglobulin (MUG), a single chain (Mr ~ 180,000) proteinase inhibitor which, like mα2M, is found at high concentrations (1 mg/ml) in adult murine plasma (21Saito A. Sinohara H. J. Biol. Chem. 1985; 260: 775-781Google Scholar, 22Abe K. Yamamoto K. Sinohara H. J. Biochem. 1989; 106: 564-568Google Scholar).Umans et al. (23Umans L. Serneels L. Overbergh L. Lorent K. Van Leuven F. Van den Berghe H. J. Biol. Chem. 1995; 270: 19778-19785Google Scholar) recently reported disruption of the mα2M gene in embryonic stem cells and the generation of homozygous α2M-deficient mice. The α2M(−/−) mice developed normally and were fertile; however, these mice responded differently from wild-type animals when challenged with certain exogenous agents. α2M(−/−) mice showed increased resistance to the lethal effects of endotoxin. We hypothesized that the endotoxin resistance of the α2M(−/−) mice might be related to the in vitro observations that hα2M neutralizes TGF-β and induces iNOS expression (14Lysiak J.J. Hussaini I.M. Webb D.J. Glass II, W.F. Allietta M. Gonias S.L. J. Biol. Chem. 1995; 270: 21919-21927Google Scholar). TGF-β suppresses expression of a number of genes associated with inflammatory reactions, including iNOS (24Vodovotz Y. Bogdan C. Paik J. Xie Q.-W. Nathan C.F. J. Exp. Med. 1993; 178: 605-613Google Scholar, 25Perrella M.A. Yoshizumi M. Fen Z. Tsai J.-C. Hsieh C.-M. Kourembanas S. Lee M.-E. J. Biol. Chem. 1994; 269: 14595-14600Google Scholar). If neutralization of TGF-β at sites of inflammation is an important function of α2M, then mα2M deficiency would allow endogenous TGF-β to function more effectively in protecting against excessive iNOS induction following endotoxin challenge.Levels of MUG in the plasma of non-pregnant α2M(−/−) mice are unchanged compared with wild-type animals (23Umans L. Serneels L. Overbergh L. Lorent K. Van Leuven F. Van den Berghe H. J. Biol. Chem. 1995; 270: 19778-19785Google Scholar), raising the question of whether MUG and mα2M are redundant as TGF-β neutralizers or NO inducers. The goals of this study were to: (i) compare the binding and neutralization of TGF-β by proteins from wild-type and α2M(−/−) mouse plasma; (ii) characterize the binding of TGF-β1 and TGF-β2 to purified mα2M and MUG; and (iii) determine whether mα2M and/or MUG promote macrophage NO synthesis. Our results demonstrate that mα2M is unique among murine α-macroglobulins, and murine plasma proteins in general, since mα2M is the only major protein that binds TGF-β and inhibits TGF-β-receptor interactions. Purified mα2M neutralized TGF-β in endothelial cell growth assays and promoted macrophage NO synthesis, while purified MUG was inactive in both cell culture systems. These studies identify TGF-β neutralization as a non-redundant activity of mα2M and suggest a mechanism for the endotoxin insensitive phenotype of α2M(−/−) mice.DISCUSSIONTGF-β functions within the context of autocrine pathways to regulate many of the properties of cells in culture, especially when the culture medium is not serum-supplemented. In colon carcinoma cells, autocrine TGF-β increases integrin α5 gene expression (44Wang D. Zhou G. Birkenmeier T.M. Gong J. Sun L. Brattain M.G. J. Biol. Chem. 1995; 270: 14154-14159Google Scholar); in rat VSMCs, autocrine TGF-β suppresses platelet-derived growth factor α-receptor expression (45Weaver A.M. Owens G.K. Gonias S.L. J. Biol. Chem. 1995; 270: 30741-30748Google Scholar) and in RAW 264.7 cells, autocrine TGF-β suppresses iNOS expression (14Lysiak J.J. Hussaini I.M. Webb D.J. Glass II, W.F. Allietta M. Gonias S.L. J. Biol. Chem. 1995; 270: 21919-21927Google Scholar). In each of these studies, the importance of autocrine TGF-β was demonstrated using a pan-isoform TGF-β-neutralizing antibody (14Lysiak J.J. Hussaini I.M. Webb D.J. Glass II, W.F. Allietta M. Gonias S.L. J. Biol. Chem. 1995; 270: 21919-21927Google Scholar, 45Weaver A.M. Owens G.K. Gonias S.L. J. Biol. Chem. 1995; 270: 30741-30748Google Scholar) or a constitutively expressed TGF-β antisense cDNA construct (46Wu S. Theodorescu D. Kerbel R.S. Willson J.K.V. Mulder K.M. Humphrey L.E. Brattain M.G. J. Cell Bio. 1992; 116: 187-196Google Scholar). Hα2M binds a number of cytokines (34Crookston K.P. Webb D.J. Wolf B.B. Gonias S.L. J. Biol. Chem. 1994; 269: 1533-1540Google Scholar); however, in the absence of exogenously added cytokines, many of the activities of hα2M in cell culture may be attributed to specific neutralization of TGF-β (14Lysiak J.J. Hussaini I.M. Webb D.J. Glass II, W.F. Allietta M. Gonias S.L. J. Biol. Chem. 1995; 270: 21919-21927Google Scholar, 45Weaver A.M. Owens G.K. Gonias S.L. J. Biol. Chem. 1995; 270: 30741-30748Google Scholar). Whether α-macroglobulins counteract TGF-β activity in vivo is undetermined.Gene knock-out experiments in mice have demonstrated that TGF-β1 functions in vivo to provide homeostatic suppression of immune/inflammatory responses (47Kulkarni A.B. Huh C.-G. Becker D. Geiser A. Lyght M. Flanders K.C. Roberts A.B. Sporn M.B. Ward J.M. Karlsson S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 770-774Google Scholar). Endotoxin challenge causes a form of inflammatory response which can be lethal. iNOS(−/−) mice are insensitive to endotoxin challenge (12MacMicking J.D. Nathan C. Hom G. Chartrain N. Fletcher D.S. Trumbauer M. Stevens K. Xie Q.-W. Sokol K. Hutchinson N. Chin H. Mudgett J.S. Cell. 1995; 81: 641-650Google Scholar), reflecting the important role played by iNOS in mediating endotoxin-associated shock (5Wolfe T.A. Dasta J.F. Ann. Pharmacother. 1995; 29: 36-46Google Scholar, 6Kilbourn R.G. Gross S.S. Jubran A. Adams J. Griffith O.W. Levi R. Lodato R.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3629-3632Google Scholar, 7Cunha F.Q. Assreuy J. Moss D.W. Rees D. Leal L.M.C. Moncada S. Carrier M. O'Donnell C.A. Liew F.Y. Immunology. 1994; 81: 211-215Google Scholar, 8Petros A. Lamb G. Leone A. Moncada S. Bennett D. Vallance P. Cardiovasc. Res. 1994; 28: 34-39Google Scholar, 9Szabo C. Mitchell J.A. Thiemermann C. Vane J.R. Br. J. Pharmacol. 1993; 108: 786-792Google Scholar). Since α2M(−/−) mice also demonstrate decreased sensitivity to endotoxin (23Umans L. Serneels L. Overbergh L. Lorent K. Van Leuven F. Van den Berghe H. J. Biol. Chem. 1995; 270: 19778-19785Google Scholar), we decided to examine the effects of the murine α-macroglobulins on TGF-β activity and iNOS activity. Although normal murine plasma has two major members of the α-macroglobulin family, only mα2M binds TGF-β1 and TGF-β2 with significant affinity. Murine α2M, but not MUG, inhibits TGF-β binding to its cellular receptors and the biological response of FBHE cells to TGF-β. Furthermore, among the murine α-macroglobulins, only mα2M induces macrophage NO synthesis. Thus, while the proteinase-inhibitory specificities of mα2M and MUG are at least partially redundant, the growth factor-carrier activities of these two proteins are not. In α2M(−/−) mice, MUG is expressed at normal levels and, during pregnancy, at increased levels (23Umans L. Serneels L. Overbergh L. Lorent K. Van Leuven F. Van den Berghe H. J. Biol. Chem. 1995; 270: 19778-19785Google Scholar); however, as shown with in vitro experiments in Fig. 1, Fig. 2, neither this protein nor any other protein in mα2M-deficient plasma substitutes for mα2M as a TGF-β carrier and neutralizer. The studies described here suggest that mα2M, in wild-type mice, may counteract TGF-β locally, at sites of inflammation, and thereby permit augmented iNOS expression in response to endotoxin. Further in vivo testing is warranted to test this hypothesis.Our analysis of NO synthesis was performed using RAW 264.7 cells. Although this is a passaged cell line, RAW 264.7 cells demonstrate excellent conservation of differentiated macrophage properties and can be primed/activated for tumorcidal and bacteriocidal activity, similarly to primary cultures of macrophages (48Haak-Frendscho M. Wynn T.A. Czuprynski C.J. Paulnock D. Clin. Exp. Immunol. 1990; 82: 404-410Google Scholar, 49Lambert L.E. Paulnock D.M. Cell. Immunol. 1989; 120: 401-418Google Scholar). Human macrophages, like the RAW 264.7 cells, secrete TGF-β (50Assoian R.K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6020-6024Google Scholar) which functions as a potent inhibitor of human macrophage activation (51Bermudez L.E. J. Immunol. 1993; 150: 1838-1845Google Scholar, 52Randow F. Syrbe U. Meisel C. Krausch D. Zuckerman H. Platzer C. Volk H.-D. J. Exp. Med. 1995; 181: 1887-1892Google Scholar). Thus, RAW 264.7 cells provide an accurate and reproducible model for studying the responses of normal macrophages.Danielpour and Sporn (41Danielpour D. Sporn M.B. J. Biol. Chem. 1990; 265: 6973-6977Google Scholar) performed experiments with serum and α2M, purified from human and bovine plasma, to demonstrate selective binding of TGF-β2, compared with TGF-β1. The KD for TGF-β1 binding to native hα2M is 30-fold higher than the KD for the TGF-β2/native hα2M interaction (34Crookston K.P. Webb D.J. Wolf B.B. Gonias S.L. J. Biol. Chem. 1994; 269: 1533-1540Google Scholar). Thus, the ability of mα2M to bind TGF-β1 and TGF-β2 with equal affinity is novel, compared with other characterized α-macroglobulins. In addition to mα2M and hα2M, we have also studied the binding of TGF-β1 to rat α1M, the constitutively synthesized homologue of hα2M, and to rat α2M, an acute-phase reactant (53Webb D.J. Crookston K.P. Figler N.F. LaMarre J. Gonias S.L. Biochem. J. 1995; 312: 579-586Google Scholar). Based on these studies, we can rank order the α-macroglobulins according to their affinities for TGF-β1 as follows: mα2M > rat α2M > rat α1M > hα2M ≫ MUG.In our analysis of TGF-β interactions with mα2M and MUG, we focused on the native forms of each protein. Activated α-macroglobulins are rapidly taken up by cells that express LRP (17Pizzo S.V. Gonias S.L. Conn P.M. The Receptors. Academic Press, Inc., New York1984: 177Google Scholar, 31Gonias S.L. Balber A.E. Hubbard W.J. Pizzo S.V. Biochem. J. 1983; 209: 99-105Google Scholar). As a result, levels of activated α-macroglobulins are negligible in the plasma under most circumstances. In the pericellular spaces, higher concentrations of activated α-macroglobulins may accumulate unless the tissue includes cells that express LRP. One concern regarding our NO synthesis experiments was whether the RAW 264.7 cells might secrete sufficient levels of proteinases to convert the α-macroglobulins into activated forms during the 24-h incubation. We previously demonstrated that this does not occur in cultures of vascular smooth muscle cells (45Weaver A.M. Owens G.K. Gonias S.L. J. Biol. Chem. 1995; 270: 30741-30748Google Scholar). To test for proteinase secretion, we incubated radiolabeled native hα2M with the RAW 264.7 cells for 24 h. The hα2M was then subjected to nondenaturing PAGE. We also measured cell-associated radioactivity and the amount of trichloroacetic acid-soluble radioactivity in the medium. None of our tests revealed detectable conversion of hα2M into the activated conformation (results not shown). Thus, the induction of NO synthesis by native mα2M and hα2M does not occur subsequent to proteinase modification of these proteins.When challenged with bleomycin, the lungs of α2M(−/−) mice were not substantially affected while the lungs of wild-type mice developed inflammatory infiltrates and early connective tissue deposition (23Umans L. Serneels L. Overbergh L. Lorent K. Van Leuven F. Van den Berghe H. J. Biol. Chem. 1995; 270: 19778-19785Google Scholar). TGF-β, which is secreted by alveolar macrophages and activated locally, has been implicated in the regulation of this process (54Denholm E.M. Rollins S.M. Amer. J. Physiol. 1993; 264: L36-L42Google Scholar). It is intriguing to speculate that the α2M(−/−) mice are bleomycin tolerant due to enhanced function of TGF-β as a general anti-inflammatory; however, TGF-β may actually promote certain aspects of the bleomycin response, such as the deposition of extracellular matrix proteins (55Giri S.N. Hyde D.M. Hollinger M.A. Thorax. 1993; 48: 959-966Google Scholar). Understanding the relationship between TGF-β, α2M, the development of immune cell infiltrates, and fibrosis, will require further investigation.In addition to its ability to neutralize TGF-β locally, α2M serves as a major carrier of TGF-β in the plasma, delaying TGF-β clearance and providing a potential pool of reversibly bound active factor (56O'Connor-McCourt M. Wakefield L.M. J. Biol. Chem. 1987; 262: 14090-14099Google Scholar, 57Huang S.S. O'Grady P. Huang J.S. J. Biol. Chem. 1988; 263: 1535-1541Google Scholar, 58Crookston K.P. Webb D.J. LaMarre J. Gonias S.L. Biochem. J. 1993; 293: 443-450Google Scholar). The unique and non-redundant activity of α2M as a TGF-β carrier in murine plasma suggests that TGF-β plasma pharmacokinetics may be altered in α2M(−/−) mice, in addition to TGF-β activity. In our cell culture systems, we have shown that mα2M induces iNOS activity in macrophages while MUG does not. These in vitro results provide a model for explaining some of the phenotypic properties of the α2M(−/−) mouse. INTRODUCTIONBacterial endotoxin is a lipopolysaccharide complex derived from the outer membranes of Gram-negative bacteria and a major mediator of the pathophysiologic changes referred to as septic shock (1Raetz C.R.H. Ulevitch R.J. Wright S.D. Sibley C.H. Ding A. Nathan C.F. FASEB J. 1991; 5: 2652-2660Google Scholar, 2Parrillo J.E. N. Engl. J. Med. 1993; 328: 1471-1477Google Scholar). Endotoxin triggers release of inflammatory cytokines, including tumor necrosis factor-α and interleukin-1β, from a number of cell types, including macrophages and endothelial cells (1Raetz C.R.H. Ulevitch R.J. Wright S.D. Sibley C.H. Ding A. Nathan C.F. FASEB J. 1991; 5: 2652-2660Google Scholar, 3Burrell R. Circ. Shock. 1994; 43: 137-153Google Scholar). These cytokines, in addition to endotoxin, induce increased cellular synthesis of nitric oxide (4Nathan C. Xie Q. J. Biol. Chem. 1994; 269: 13725-13728Google Scholar, 5Wolfe T.A. Dasta J.F. Ann. Pharmacother. 1995; 29: 36-46Google Scholar, 6Kilbourn R.G. Gross S.S. Jubran A. Adams J. Griffith O.W. Levi R. Lodato R.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3629-3632Google Scholar, 7Cunha F.Q. Assreuy J. Moss D.W. Rees D. Leal L.M.C. Moncada S. Carrier M. O'Donnell C.A. Liew F.Y. Immunology. 1994; 81: 211-215Google Scholar). NO 1The abbreviations used are: NOnitric oxideNOSnitric oxide synthaseiNOSinducible nitric oxide synthasehα2Mhuman α2-macroglobulinmα2Mmurine α2-macroglobulinMUGmurinoglobulinTGF-βtransforming growth factor-βLRPlow density lipoprotein receptor-related proteinFBSfetal bovine serumBS3bis(sulfosuccinimidyl) suberateFBHEfetal bovine heart endothelialPAGEpolyacrylamide gel electrophoresis. plays a significant role in mediating the profound vasodilation, hypotension, and hyporeactivity to pressor agents, which are characteristic of severe septic shock (2Parrillo J.E. N. Engl. J. Med. 1993; 328: 1471-1477Google Scholar, 5Wolfe T.A. Dasta J.F. Ann. Pharmacother. 1995; 29: 36-46Google Scholar, 6Kilbourn R.G. Gross S.S. Jubran A. Adams J. Griffith O.W. Levi R. Lodato R.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3629-3632Google Scholar, 7Cunha F.Q. Assreuy J. Moss D.W. Rees D. Leal L.M.C. Moncada S. Carrier M. O'Donnell C.A. Liew F.Y. Immunology. 1994; 81: 211-215Google Scholar, 8Petros A. Lamb G. Leone A. Moncada S. Bennett D. Vallance P. Cardiovasc. Res. 1994; 28: 34-39Google Scholar, 9Szabo C. Mitchell J.A. Thiemermann C. Vane J.R. Br. J. Pharmacol. 1993; 108: 786-792Google Scholar).NO is produced by three enzymes of the nitric oxide synthase (NOS) family (4Nathan C. Xie Q. J. Biol. Chem. 1994; 269: 13725-13728Google Scholar, 10Nathan C. Xie Q.-W. Cell. 1994; 78: 915-918Google Scholar). Two constitutive forms of NOS (NOS-I, NOS-III) produce low levels of NO in response to agonists which increase intracellular calcium. The resulting NO is important in the regulation of normal homeostatic processes, including blood pressure and neuronal transmission (10Nathan C. Xie Q.-W. Cell. 1994; 78: 915-918Google Scholar). The inducible form of NOS (iNOS or NOS-II) produces high levels of NO, independently of intracellular calcium. The NO produced by iNOS functions in the immune response to pathogens and may also provide cytotoxic activity toward cancer cells (10Nathan C. Xie Q.-W. Cell. 1994; 78: 915-918Google Scholar). Since iNOS activity is not calcium-dependent, regulation of iNOS expression is critical and many cytokines are involved in this process in various cell types (11De Vera M.E. Shapiro R.A. Nussler A.K. Mudgett J.S. Simmons R.L. Morris S.M. Billiar T.R. Geller D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1054-1059Google Scholar).Intravenous administration of endotoxin in experimental animals causes an initial, transient increase in NO that is probably due to stimulation of constitutive NOS followed by a sustained increase in NO produced by iNOS (8Petros A. Lamb G. Leone A. Moncada S. Bennett D. Vallance P. Cardiovasc. Res. 1994; 28: 34-39Google Scholar). Mice that are iNOS-deficient, due to gene disruption in embryonic stem cells, demonstrate resistance to doses of endotoxin that are lethal in wild-type animals (12MacMicking J.D. Nathan C. Hom G. Chartrain N. Fletcher D.S. Trumbauer M. Stevens K. Xie Q.-W. Sokol K. Hutchinson N. Chin H. Mudgett J.S. Cell. 1995; 81: 641-650Google Scholar, 13Wei X.Q. Charles I.G. Smith A. Ure J. Feng G.-J. Huang F.P. Xu D. Muller W. Moncada S. Liew F.Y. Nature. 1995; 375: 408-411Google Scholar). iNOS(−/−) mice are also less effective at eliminating infections caused by Listeria monocytogenes or Leishmania major. Macrophages that are isolated from iNOS(−/−) mice are less effective killers of cancer cells in vitro (12MacMicking J.D. Nathan C. Hom G. Chartrain N. Fletcher D.S. Trumbauer M. Stevens K. Xie Q.-W. Sokol K. Hutchinson N. Chin H. Mudgett J.S. Cell. 1995; 81: 641-650Google Scholar).Human α2-macroglobulin (hα2M) is a proteinase inhibitor and specific cytokine carrier that induces iNOS expression and NO synthesis in the murine macrophage-like cell line, RAW 264.7 (14Lysiak J.J. Hussaini I.M. Webb D.J. Glass II, W.F. Allietta M. Gonias S.L. J. Biol. Chem. 1995; 270: 21919-21927Google Scholar). The mechanism of iNOS induction involves the neutralization of autocrine cytokine activity by hα2M. Since RAW 264.7 cells synthesize, secrete, and activate transforming growth factor-β (TGF-β) and because TGF-β-neutralizing antibody induces RAW 264.7 cell NO synthesis, similarly to hα2M, we proposed that neutralization of autocrine TGF-β is most likely responsible for the increase in iNOS expression in hα2M-treated RAW 264.7 cells (14Lysiak J.J. Hussaini I.M. Webb D.J. Glass II, W.F. Allietta M. Gonias S.L. J. Biol. Chem. 1995; 270: 21919-21927Google Scholar).Hα2M exists in at least two well defined conformations (15Barrett A.J. Starkey P.M. Biochem. J. 1973; 133: 709-724Google Scholar, 16Gonias S.L. Reynolds J.A. Pizzo S.V. Biochim. Biophys. Acta. 1982; 705: 306-314Google Scholar). The native form is active as a proteinase inhibitor, but not recognized by the α2M receptor, low density lipoprotein receptor-related protein (LRP) (17Pizzo S.V. Gonias S.L. Conn P.M. The Receptors. Academic Press, Inc., New York1984: 177Google Scholar). Thus, native α2M is stable in the blood and interstitial spaces. The activated form of α2M, generated by reaction with proteinase, is LRP-recognized. In the RAW 264.7 cell system, native α2M and activated α2M are comparably effective as inducers of NO synthesis (14Lysiak J.J. Hussaini I.M. Webb D.J. Glass II, W.F. Allietta M. Gonias S.L. J. Biol. Chem. 1995; 270: 21919-21927Google Scholar).The murine α-macroglobulin family includes at least five distinct genes, two of which encode for proteins that have been purified and characterized (18Overbergh L. Torrekens S. Van Leuven F. Van den Berghe H. J. Biol. Chem. 1991; 266: 16903-16910Google Scholar, 19Van Leuven F. Torrekens S. Overbergh L. Lorent K. De Strooper B. Van den Berghe H. Eur. J. Biochem. 1992; 210: 319-327Google Scholar, 20Overbergh L. Hilliker C. Lorent K. Van Leuven F. Van den Berghe F. Genomics. 1994; 22: 530-539Google Scholar). Murine α2M (mα2M) is a tetrameric protein (Mr ~ 720,000) and a close homologue of human α2M. Hα2M and mα2M share comparably broad proteinase-inhibitory specificities and undergo equivalent proteinase-induced conformational changes. The second member of the murine α-macroglobulin family is murinoglobulin (MUG), a single chain (Mr ~ 180,000) proteinase inhibitor which, like mα2M, is found at high concentrations (1 mg/ml) in adult murine plasma (21Saito A. Sinohara H. J. Biol. Chem. 1985; 260: 775-781Google Scholar, 22Abe K. Yamamoto K. Sinohara H. J. Biochem. 1989; 106: 564-568Google Scholar).Umans et al. (23Umans L. Serneels L. Overbergh L. Lorent K. Van Leuven F. Van den Berghe H. J. Biol. Chem. 1995; 270: 19778-19785Google Scholar) recently reported disruption of the mα2M gene in embryonic stem cells and the generation of homozygous α2M-deficient mice. The α2M(−/−) mice developed normally and were fertile; however, these mice responded differently from wild-type animals when challenged with certain exogenous agents. α2M(−/−) mice showed increased resistance to the lethal effects of endotoxin. We hypothesized that the endotoxin resistance of the α2M(−/−) mice might be related to the in vitro observations that hα2M neutralizes TGF-β and induces iNOS expression (14Lysiak J.J. Hussaini I.M. Webb D.J. Glass II, W.F. Allietta M. Gonias S.L. J. Biol. Chem. 1995; 270: 21919-21927Google Scholar). TGF-β suppresses expression of a number of genes associated with inflammatory reactions, including iNOS (24Vodovotz Y. Bogdan C. Paik J. Xie Q.-W. Nathan C.F. J. Exp. Med. 1993; 178: 605-613Google Scholar, 25Perrella M.A. Yoshizumi M. Fen Z. Tsai J.-C. Hsieh C.-M. Kourembanas S. Lee M.-E. J. Biol. Chem. 1994; 269: 14595-14600Google Scholar). If neutralization of TGF-β at sites of inflammation is an important function of α2M, then mα2M deficiency would allow endogenous TGF-β to function more effectively in protecting against excessive iNOS induction following endotoxin challenge.Levels of MUG in the plasma of non-pregnant α2M(−/−) mice are unchanged compared with wild-type animals (23Umans L. Serneels L. Overbergh L. Lorent K. Van Leuven F. Van den Berghe H. J. Biol. Chem. 1995; 270: 19778-19785Google Scholar), raising the question of whether MUG and mα2M are redundant as TGF-β neutralizers or NO inducers. The goals of this study were to: (i) compare the binding and neutralization of TGF-β by proteins from wild-type and α2M(−/−) mouse plasma; (ii) characterize the binding of TGF-β1 and TGF-β2 to purified mα2M and MUG; and (iii) determine whether mα2M and/or MUG promote macrophage NO synthesis. Our results demonstrate that mα2M is unique among murine α-macroglobulins, and murine plasma proteins in general, since mα2M is the only major protein that binds TGF-β and inhibits TGF-β-receptor interactions. Purified mα2M neutralized TGF-β in endothelial cell growth assays and promoted macrophage NO synthesis, while purified MUG was inactive in both cell culture systems. These studies identify TGF-β neutralization as a non-redundant activity of mα2M and suggest a mechanism for the endotoxin insensitive phenotype of α2M(−/−) mice.
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