Jan 3, 1990 - by Thomas. (28). Following ...... technical assistance and to Tom Sanford for help in construction of cytokine ... C. A.. Punsal. P. I.. and Colten. H.
THE JOURNAL OF BIOLOGICAL CHEMLWRY 8 1990 by The American Society for Biochemistry
Endotoxin
Induction
Vol. 26.5, No. 25, Issue of September 5, pp. 15267-15274.1990
Printed in U.S. A.
and Molecular Biology, Inc.
of Murine
Metallothionein
Gene Expression* (Received
Swapan From
K. De&
the Department
Michael T. McMasteri, and Glen K. Andrewsll of Biochemistry and Molecular Biology, University of Kansas
Bacterial endotoxin-lipopolysaccharide (LPS) rapidly induced hepatic metallothionein (MT) mRNA levels in the LPS-sensitive CD- 1 strain of mice. This LPS effect was severely attenuated in the LPS-resistant C3H/HeJ strain of mice, but could be mimicked by injection of human recombinant interleukin-la (IL- la) or human recombinant tumor necrosis factor (TNF-(r). In the CD-l strain, LPS induction of MT gene expression occurred in each of 10 organs examined (liver, kidney, pancreas, intestine, lung, heart, brain, ovary, uterus, and spleen). Solution hybridization with probes specific for MT-I or MT-II mRNA established that these genes were co-induced in each of the organs and that the liver and kidney contained the highest absolute levels of these mRNAs, whereas in the intestine and spleen they were lo-20-fold lower. LPS and cytokine induction of hepatic MT gene expression occurred in hypophysectomized mice, which suggests a lack of significant involvement of glucocorticoids. Several recombinant cytokines (TNF-a, IL-la, IL-la, IL-6, interferon-y (IFN--y), as well as poly(rI.rC) were effective inducers of hepatic MT-I and MT-II genes. As an attempt to determine which of these cytokines may mediate LPS effects on MT gene expression in UILJO, CD-1 mice were injected with LPS or various cytokines, and RNA from liver, ovary, and uterus was extracted at various times postinjection and analyzed by Northern blotting using probes specific for IL-la, IL-lj3, TNF-a, IL-6, and MT mRNA. In each organ examined, LPS, IL-la, or IL-l/3 injection caused a rapid, coordinate, transient increase in the levels of each of the cytokine mRNAs which peaked by 1 h and declined to low levels by 4 h. In contrast, levels of MT mRNA did not reach a peak until 4-6 h postinjection. TNF-a had minimal effects on expression of cytokine and MT genes in organs other than liver. IL-6 had no effect on hepatic cytokine mRNA levels, and induced MT mRNA only in the liver which suggests a direct effect of IL-6 on hepatic MT gene expression. These data suggest that the acute effects of LPS on MT gene expression may include complex paracrine interactions between a variety of cytokines and the cells expressing MT genes in each organ, and tissue-specific cytokine effects on the MT genes. * This work was supported in part by National Institute of Health Grant ES 04725 and United States Department of Agriculture Grant 88-37266-4117 (to G. K. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Funded by a Wesley Foundation Scholar Program in Cancer Research postdoctoral fellowship. § A March of Dimes predoctoral fellow. V To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, WHE 4018, University of Kansas Medical Ctr., 39th and Rainbow Blvd., Kansas City, KS 66103. Tel.: 913588-6935; Fax: 913-588-5677.
Medical
for publication,
Center,
Kansas
January
City,
Kansas
3, 1990)
66103
Metallothioneins (MTs)’ are a class of small, cysteine-rich, heavy metal binding proteins. In the mouse, two isoforms of MT (MT-I and MT-II), which differ slightly in amino acid sequence and net charge, are encoded in the genome (1).
Expression of mouse MT genes can be up-regulated in response to a variety of stresses including inflammation (for reviews see Refs. 2-4). The observation that hepatic expression of MT is dramatically elevated in response to bacterial infection, an effect mediated by endotoxin-lipopolysaccharide (LPS) (5), has lead to the classification of MT as an acutephase protein (6). This effect of LPS on hepatic MT gene expression has been conserved during evolution (7, 8). It occurs independent of glucocorticoid hormones (9,lO) and is, at least in part, dependent on the LPS-induced activation of macrophages since this response is attenuated in the LPSresistant C3H/HeJ strain of mice (11). The macrophagederived factor IL-la has been shown to induce MT gene expression in cultured cells (12) and following injection in uiuo (10, 13, 14), as has IFN-a (15-17). Although the effects of IL-101 on the acute-phase response are well documented (18), it is equally apparent that TNF-cu can elicit many of the biological effects attributed to IL-l (19, 20), including the induction of the acute-phase proteins (21). Furthermore, recent evidence implicates IL-6 as a major regulator of the acute-phase response both in viva and in uitro (22, 23). It is also apparent that these cytokines can have synergistic effects (24) and increase each others synthesis (20). The potential involvement of other cytokines in mediating LPS effects on MT gene expression has not been previously reported. In this study the acute effects of LPS and various cytokines on MT gene expression were analyzed at the level of mRNA. It was found that LPS induces acute increases in MT mRNA levels in a wide variety of organs. In those organs examined (pancreas, ovary, uterus) this increase was preceded by increased expression of IL-la and -& TNF01, and IL-6 genes as measured by changes in mRNA levels. An injection of any of several recombinant cytokines (IL-la, IL-l& IL-6, TNF-LU, IFN-7) was also shown to induce MT gene expression, but the effects of IL-6 and TNF-a were restricted to the liver. EXPERIMENTAL
PROCEDURES
Animals-Female CD-1 outbred mice (Charles River Laboratories) and female C3H/HeJ inbred mice (Jackson Laboratories) were housed in the animal care facility at the University of Kansas Medical Center. Animals were injected intraperitoneally with (100 ~1) of phosphate-buffered saline (PBS) containing LPS or cytokines or with PBS alone. Cytokines were diluted fresh to appropriate concentrations in sterile PBS and injected immediately. Tissue samples were removed at various times after injection, as specified in the text, and ’ The abbreviations used are: MT, metallothionein: IFN, interferon; IGF, insulin-like growth factor; IL-l, interleukin-1; I IL-6 > TNF-ol. An injection of IL-la (Fig. 6B) increased hepatic MT mRNA as expected, and this induction was clearly detectable by 1 h, and peaked by 4-6 h postinjection. However, IL-la, IL-lp, IL-6, and TNF-o( mRNAs were also induced, and coordinately reached maximal levels by 1 h postinjection, after which they declined (Fig. 6B). The IL-6 blot in Fig. 6B depicts two hybridization signals. The upper band co-migrated with 18 S ribosomal RNA, was only detected following lengthy autoradiographic exposures, and was preferentially eliminated by increasing the stringency of the hybridization (data not shown). Whether this represents hybridization to an IL-6 related transcript or nonspecific hybridization remains to be resolved. The relative level of induction of these cytokine
Injection
B Liver
Uterus
IL- 1 ot mRNA 0
9
W
Injection FIG. 7. The acute induction of IL-lb and IL-la mRNAs in the liver, ovary, and uterus following an injection of IL-la, IL-lb, TNF-a, IL-6, or LPS. RNA was prepared from the liver, ovary, and uterus of female CD-l mice 1 h following an intraperitoneal injection of LPS from E. coli Oll:B4 (100 rg) or the recombinant cytokines human IL-lo (1 pg; 3 X 10” units), mouse IL-l/3 (1 pg; 1 X 10” units), human IL-6 (5 ~.rg; 2 x 10’ units), and human TNF-n (1 Kg; 2 x 10” units). RNA (6 pg) was analyzed by Northern blot hybridization using a cRNA probe for mouse IL-l/3 (A) or IL-ltu (B). RNA (2 pg) from a mouse macrophage cell line (J774A.l) stimulated with LPS (1 rg/ml) for 4 h (A40) was also Northern blotted as a comparative standard.
15272
Endotoxin
Regulation
TABLE III Metallothionein-I injection
and -II mRNA leveki of LPS, recombinant Ovary
Treatmenta
MT
Isoformb
in ovary and uterus cytokines, and zinc
following
uterus MT Isoform*
I I II II Zinc 4.1 4.4 2.6 2.3 LPS 1.5 9.4 9.9 7.3 IL-lol 3.0 3.3 6.4 6.0 IL-l@ 3.5 5.3 7.6 6.6 IL-6 1.2 1.5 1.0 1.1 TNF-a 1.1 1.2 1.2 1.2 a RNA samples were obtained 4 h following an injection of zinc (100 pmol/kg), LPS (100 fig), or the recombinant csokines human IL-la (1 ug: -_ 3 X lo5 units). mouse IL-W (1 ua: 1 X lo5 units). human IL-6 (5 pg; 2 x lo4 units),‘and human TNF:g’(l pg; 2 x 105’units). * Data represent -fold increase in MT-I and MT-II mRNA levels relative to those in control (untreated) ovary or uterus determined by oligodeoxyribonucleotide excess solution hybridization. 18, or LPS in the liver, ovary, and uterus. TNF-(Y effectively induced hepatic expression of IL-18 and IL-la mRNAs, which peaked by 1 h postinjection. However, TNF-a had reduced effects on IL-lfi mRNA, and little or no effect on IL-la mRNA in the ovary and uterus relative to those of injected LPS and IL-la or IL-l& IL-6 did not effect the level of ILl/3 or IL-la mRNAs (Fig. 7) or the other cytokine mRNAs in liver, ovary, or uterus (data not shown). Thus, each of these cytokines tested can rapidly induce hepatic MT mRNA, but IL-la, IL-l& and to a lesser extent TNF-(w, each in turn, rapidly effected the expression of the other cytokine genes in the liver. However, only those agents (LPS, IL-la, and ILlp) which induced substantial changes in several cytokine mRNA levels in the ovary and uterus (and pancreas; data not shown) were effective inducers of MT mRNA in those organs. These data suggest that the acute effects of LPS on MT gene expression may, in part, involve complex paracrine interactions between a variety of cytokines, and the cells expressing MT in each organ. In contrast, IL-6 apparently exerts direct effects specifically on hepatic MT gene expression. DISCUSSION MTs are intracellular heavy metal binding proteins (for reviews see Refs. 2-4) which are induced as a component of the acute-phase response (5,6,34) and serve to sequester zinc from the serum (14). Although LPS is known to induce MT mRNAs in the mouse liver, kidney, heart, and brain (9), and IL-la injection induces MT mRNAs in rat liver, marrow, and thymus (14), this study is the first to demonstrate that an acute inductive effect of bacterial endotoxin (LPS) on both the MT-I and the MT-II gene occurs in a wide variety of organs. In addition, it was shown for the first time that the hepatic induction of these genes, as measured by acute changes in MT mRNA levels, occurs in response to injection of several recombinant cytokines (IL-la, IL-l& IL-6, TNFcy, INF-r). The rapid induction of MT genes by LPS in such a wide range of organs suggests that altered zinc metabolism, in response to inflammatory stress, is not simply the result of selective tissue-specific increases in MT as has been previously suggested (14). However, it is also clear from these studies that the levels of MT mRNAs in liver are the highest among the organs examined which is consistent with a central role for this organ in zinc metabolism. One interesting exception was the testes which has constitutively high levels of MT mRNA which are unaltered by LPS injection’ as has been ’ S. K. De, G. Enders, and G. K. Andrews, unpublished
data.
of Metallothionein
Genes
previously demonstrated in the mouse (9, 35). The ability of the MT genes in many organs to respond to LPS suggests that local inflammation or release of cytokines can lead to increased MT synthesis selectively in the proximal tissues. Whether this is true for other acute-phase proteins remains to be determined. However, several other acute-phase proteins in the rat are apparently induced in a tissue-specific manner
(36). Induction of the mouse MT genes by LPS is rapid and results in a IO-20-fold increase in mRNA levels by 4 h postinjection. During the inflammatory response of acutephase proteins in the rat liver, rapid changes in the levels of mRNAs for cuz-macroglobulin and fibrinogen occur, whereas several other mRNAs show a delayed increase (36). Both the magnitude and the rapidity of the response of the MT genes to LPS suggest that MT is a major component of the acutephase response. In several experiments it was noted that the MT mRNA increased in size as well as abundance following induction by LPS or other cytokines. In the rat, al-acid glycoprotein mRNA is longer immediately after induction, and this is due to altered trimming of the poly(A) tail (36). Whether this accounts for the changes in MT mRNA length remains to be determined. However, a progressive reduction in the size of the poly(A) tail of rat MT-I mRNA following induction by copper or dexamethasone has been reported (37). An interesting observation made during the course of these studies is that IGF-I mRNA is significantly reduced in the livers of LPS or cytokine treated mice. There are several other well documented negative acute-phase proteins (albumin, a-globulin, transthyretin) whose transcription rates are decreased following acute inflammation (36). Hepatic IGF-I mRNA is regulated by growth hormone release from the anterior pituitary (32), and during inflammation release of growth hormone is increased perhaps by a direct effect of cytokines on the pituitary (30-40). The decrease in hepatic IGF-I mRNA, therefore, likely results from a direct effect of cytokines on the liver cells leading to decreased transcription of the IGF-I gene or destabilization of this mRNA. It is now well documented that LPS initiates the acutephase response, in part, by the activation of macrophages which then produce several cytokines such as IL-la, IL-l& IL-6, and TNF-(Y. The lack of LPS sensitivity in the C3H/ HeJ mouse reflects a severely reduced macrophage activation in response to LPS. In these mice, LPS injection failed to induce significant accumulation of hepatic MT mRNA which accounts for the previously reported attenuation of MT protein induction (11). However, injection of TNF-a or IL-la effectively induced hepatic MT mRNA in these mice. The observation that IL-la can induce MT gene expression in some cultured human cells (12), and following injection into rats (10, 13, 14), has led to the widespread belief that this cytokine is of primary importance in regulating MT gene expression during the acute-phase response. However, IFN-(Y is also an effective inducer of human MT (15-17), and this study documented, for the first time, the induction of MT genes in uivo by exogenous IL-l& IL-6, TNF-cu, and IFNs, as well as IL-la. It has been suggested previously that the acute-phase response may he regulated in uiuo by the concerted actions of several cytokines produced by the macrophage, as well as by other cells (23, 34). The results of this study support that hypothesis. Following an injection of LPS, increased MT mRNA levels in several organs (liver, ovary, pancreas, uterus) were preceded or accompanied by a coordinate induction of the IL-la and -p, IL-6, and TNF-(U genes. This suggests that the local release of these cytokines may regulate the initial
Endotoxin
Regulation of Metallothionein
induction of the MT genes. Although the cell type(s) expressing these cytokine genes in each organ was not determined, the coordinated increase in cytokine gene expression is consistent with a common cellular origin, i.e. the macrophage. However, the perpetuation and amplification of the acutephase response likely involves cytokine release by a variety of other cell types (34). That the increased levels of cytokine mRNAs resulted in increased synthesis, processing, and release of active cytokine was not documented in these experiments, but it has been shown previously that administration of endotoxin or cytokines leads to rapidly increased (within minutes) serum levels of TNF-cu (19, 41, 42) and IL-6 (43), whereas IL-l is apparently not rapidly elevated to high levels in the serum (34), but instead increases slowly (42). The finding that LPS rapidly and dramatically induced IL-
la and -p mRNAs in several organs suggests that these cytokines exert their effects initially through a paracrine mechanism. The relative levels of these mRNAs (IL-la and -/3) were greater than those of TNF-a and IL-6. IL-l/3 mRNA
was the most abundant, and it has been reported previously that IL-18 represents l-2% of the macrophage mRNA following LPS stimulation (20). The finding amounts of this cytokine induced
that injection of modest MT mRNA in several
organs, coupled with the high abundance of this mRNA following LPS stimulation, suggests a central role for IL-l/3 in the regulation of MT gene expression. A possible intrago-
nadal autocrine/paracrine function for IL-l in the ovary has also been suggested, and IL-l/3 is much more potent than ILla in suppressing
follicle-stimulating
hormone-induced
gran-
ulosa cell differentiation (i.e. luteinizing hormone receptor development and progesterone synthesis) (44). Thus, our data suggest that IL-lb exerts paracrine effects on expression of several genes in the ovary. However, it was noted that injection of IL-la or -8, in turn, induced the rapid coordinate expression of the other cytokine genes, and in the ovary and uterus increased levels of IL-lp mRNA alone did not ensure induction
of the MT genes. This further
been shown to rapidly
induce
IL-6
levels in the serum
MT genes. To examine
further
the regulation
of
by cytokines,
in vitro models
will
be
Acknowledgments-We are indebted to Cathy O’Rourke for expert technical assistance and to Tom Sanford for help in construction of cytokine riboprobe vectors. The mouse macrophage cell line J774A.l was provided
by Dr. Tsuneo
Suzuki,
Department
of Microbiology
and
Immunology, University of Kansas Medical Center. We would also like to acknowledge the helpful discussions and contributions of Drs. Gary Wood, David Morrison, and Steven Russell. REFERENCES
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Therefore, the possibility exists that TNF-LU exerts its effects on hepatic MT gene expression indirectly via induction of IL6. A lack of TNF-a effects on MT mRNA levels in organs other than liver further suggests a lack of significant involvement of circulating IL-6 in regulation of extrahepatic MT genes. Although we cannot exclude the possibility that rapid clearance of IL-6 by the liver prevents MT induction in other organs, the IL-6 bound to the liver following injection is not degraded but is subsequently released (45), and injection of TNF-a can lead to elevated serum IL-6 levels two h later (43). In conclusion, the data reported here suggest that endotoxin regulation of mouse MT genes is a complex process involving paracrine and tissue-specific effects of several cytokines. An important role for IL-la and -p in regulation of extrahepatic MT genes is suggested, as is a direct role for IL-6 in regulation of hepatic
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suggests a cooperative
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