- Open Access
Lipopolysaccharide-mediated signal transduction: Stabilization of TNF-alpha mRNA contributes to increased lipopolysaccharide-stimulated TNF-alpha production by Kupffer cells after chronic ethanol feeding
© Kishore et al; licensee BioMed Central Ltd 2004
- Published: 14 January 2004
- Kupffer Cell
- Alcoholic Liver Disease
- Ethanol Exposure
- Chronic Ethanol
- Ribonuclease Protection Assay
Alcoholic liver disease (ALD) develops in approximately 20% of all alcoholics with a higher prevalence in females . The development of fibrosis and cirrhosis is a complex process involving both parenchymal and non-parenchymal cells resident in the liver, as well as the recruitment of additional cell types to the liver in response to damage and inflammation . Kupffer cells, the resident macrophages in the liver, are critical to the onset of ethanol-induced liver injury. Ablation of Kupffer cells prevents the development of fatty liver and inflammation, early events in the progression of ethanol-induced liver damage, in rats chronically exposed to ethanol via intragastric feeding . Endotoxin (or lipopolysaccharide (LPS)), a component of the cell wall of gram-negative bacteria, is an important activator of Kupffer cells, stimulating the production of inflammatory and fibrogenic cytokines, as well as reactive oxygen species. LPS concentration is increased in the blood of alcoholics [4, 5] and rats exposed to ethanol via intra-gastric infusion , probably due to impaired barrier function of the intestinal mucosa ). In a series of elegant experiments using transgenic animals from the laboratory of Ron Thurman, a working model for the development of alcoholic liver disease has been developed. This model proposes that increased exposure of Kupffer cells to LPS during chronic ethanol consumption results in increased production of inflammatory mediators, in particular TNF-alpha and reactive oxygen species, leading to the progression of fatty liver, inflammation and fibrosis, characteristic of ALD . In addition to this increased exposure of Kupffer cells to LPS in response to ethanol, we and others have shown that chronic ethanol also sensitizes Kupffer cell responses to LPS [8, 9]. We hypothesize that increased sensitivity to LPS stimulation after chronic ethanol exposure likely contributes to the progression of liver injury.
Role of TNF-alpha in the progression of alcoholic liver disease
TNF-alpha is thought to play a particularly critical role in the pathogenesis of ALD. TNF-alpha is one of the principal mediators of the inflammatory response in mammals, transducing differential signals that regulate cellular activation and proliferation, cytotoxicity and apoptosis [10, 11] In addition to its role in acute septic shock, TNF-alpha has been implicated in the pathogenesis of a wide variety of inflammatory diseases [11, 12, 14] as well as in the progression of alcoholic liver disease [7, 15] The role of TNF-alpha in the development of ethanol-induced liver injury has been well characterized in animal models [7, 15]
Production of TNF-alpha is one of the earliest responses of the liver to injury . Circulating TNF-alpha is increased in the blood of alcoholics and in animals chronically exposed to ethanol [16, 17]. Antibiotic treatment decreases TNF-alpha expression and ethanol-induced liver injury in rats exposed to ethanol via intra-gastric infusion , suggesting that increased TNF-alpha after ethanol exposure is due, at least in part, to increased exposure to LPS. In addition to increasing LPS exposure, chronic ethanol also increases sensitivity to LPS. For example, long-term ethanol consumption increases the susceptibility of rats to endotoxin-induced liver injury [8, 18]. Moreover, we have shown that LPS-stimulated TNF-alpha secretion is increased in Kupffer cells isolated from rats fed ethanol in their diet for 4 weeks compared to pair-fed controls [9, 19, 20].
Regulation of LPS-stimulated TNF-alpha production
Ethanol disrupts a number of hormone and neurotransmitter dependent signaling pathways , including many of the same signaling pathways activated by LPS in macrophages. In a series of recent experiments, we have found that chronic ethanol feeding disrupts specific LPS-stimulated signal transduction pathways which regulate both TNF-alpha transcription and mRNA stability in Kupffer cells [19, 20, 30]. Chronic ethanol had complex effects on the regulation of LPS-stimulated TNF-alpha mRNA transcription; the transcriptional activity of NF kappa B was dramatically decreased, but this was compensated for by increased Egr-1 activity [19, 30]. Despite these complex changes, chronic ethanol exposure had no net effect on the rate of TNF-alpha transcription . Therefore, we hypothesized that increased LPS-stimulated TNF-alpha mRNA accumulation in Kupffer cells isolated from rats chronically exposed to ethanol might be due to a stabilization of TNF-alpha mRNA.
Chronic ethanol feeding protocol
Male Wistar rats (150 g) were allowed free access to liquid diet  with 17% of calories as ethanol for 2 days and then provided with diet containing 35% of the calories from ethanol for 4 weeks. Controls were pair-fed a liquid diet that was identical to the ethanol diet except that maltose dextrins were isocalorically substituted for ethanol . Procedures involving animals were approved by the Institutional Animal Care Board at Case Western Reserve University.
Kupffer cell isolation and culture
Kupffer cells were isolated as previously described  except that CMRL media was used to isolate and culture Kupffer cells. Briefly, livers were perfused with 0.05% collagenase and the resulting suspension of liver cells treated with 0.02% pronase for 15 min at 12 degrees C. The cell suspension was centrifuged 3 times at 50 – g for 2 min and the supernatant collected after each centrifugation. The pooled supernatant was then centrifuged at 500 – g for 7 min to collect non-parenchymal cells. Kupffer cells were then purified by centrifugal elutriation .
Isolated Kupffer cells were suspended in CMRL with 10% fetal bovine serum and penicillin-streptomycin at a concentration of 2 – 106 cells/ml and plated onto 96 well (0.2 ml/well), 6-well (3 ml/well) or 100 mm plates (5 ml/well) culture plates. After 2 h, non-adherent cells were removed by aspiration and fresh media supplied. Assays were initiated after 20–24 h in culture.
Culture and chronic ethanol exposure of RAW264.7 macrophages
The mouse macrophage-like RAW264.7 cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD). Cells were cultured in DMEM supplemented with 10% FBS and penicillin-streptomycin at 37 degrees C in a 5% CO2 atmosphere. For ethanol treatments, cells were incubated with 25 mM ethanol for 48 h; culture dishes were wrapped in parafilm to minimize the evaporation during culture. Culture dishes with untreated cells were also wrapped in parafilm.
Ribonuclease protection assay for TNF-alpha mRNA
After 20 h in culture, Kupffer cell media was removed and replaced with fresh media containing 10% fetal bovine serum. Cells were then stimulated with 0 or 100 ng/ml LPS for 1 h. In some experiments, cells were pre-incubated with PD98059, SB20380 or vehicle (DMSO) for 2 h prior to LPS stimulation, or treated with 5 micrograms/ml actinomycin D 1 h after LPS stimulation and then harvested after 1–2 h. Total RNA was isolated by the TRIzol method (Gibco, Grand Island, NY). Rat cytokine multiprobe DNA templates (Pharmingen, San Diego, CA) were used to synthesize in vitro transcribed anti-sense riboprobes and ribonuclease protection assays were carried out following manufacturer's instructions. Samples were run on 5% sequencing gels, dried and autoradiographed.
Chronic ethanol increases LPS-stimulated TNF– expression
Kupffer cells isolated from rats fed ethanol for 4 weeks accumulated 2-fold higher concentration of TNF-alpha in their media in response to challenge with LPS, with no difference in TNF-alpha accumulation between the pair- and ethanol-fed groups when cells were not treated with LPS (basal) [9, 19]. Increased accumulation of bioactive TNF-alpha after chronic ethanol was associated with increased accumulation of TNF-alpha mRNA in response to LPS [19, 20].
Increased LPS-stimulated TNF alpha production by Kupffer cells after chronic ethanol feeding could be due to a direct effect of chronic ethanol exposure on the Kupffer cell itself or to a more systemic response, involving other tissues and organs sensitive to ethanol. If chronic ethanol acts directly on the Kupffer cell, then exposure of macrophages to ethanol during in vitro culture should mimic the in vivo response. RAW264.7 macrophages were cultured with and without 25 mM ethanol for 48 h and then stimulated or not with 100 ng/ml LPS for 2 to 4 h. While LPS increased TNF-alpha production in control cells, secretion of TNF-alpha in response to LPS was increased by 1.7–2.2-fold in cells cultured with ethanol compared to controls . Increased LPS-stimulated TNF-alpha secretion after chronic ethanol exposure was associated with increased TNF-alpha mRNA accumulation .
Taken together, these data indicate that there is a strong similarity between the effects of ethanol feeding on Kupffer cell responses to LPS and the in vitro response of RAW 264.7 macrophages to culture with chronic ethanol. These results suggest that changes in TNF-alpha production by macrophages observed after long term ethanol exposure in vivo are not due solely to systemic responses, such as increased exposure to endotoxin/LPS [4–6] or changes in retinoic acid status [5, 6, 33], but are due, at least in part, to a direct effect of ethanol exposure on macrophage function.
LPS- induced TNF-alpha mRNA is stabilized by chronic ethanol exposure
While LPS-induced TNF-alpha production is controlled at transcriptional, post-transcriptional and post-translational levels , increased transcription is the initial response to LPS. Therefore, we investigated whether chronic ethanol affected LPS-stimulated TNF-alpha transcription. Kupffer cells isolated from pair- and ethanol-fed rats, as well as RAW 264.7 macrophages cultured with or without 25 mM ethanol, were stimulated with 0 or 100 ng/ml LPS, nuclei isolated and used for run-on transcription assays. We found that chronic ethanol exposure, either in vivo or during culture, had no net effect on TNF-alpha transcription .
Since chronic ethanol was not acting to increase transcription of TNF-alpha, we hypothesized that chronic ethanol-induced increases in LPS-stimulated TNF-alpha mRNA accumulation might reflect an increased half-life of the transcripts. To test this hypothesis, mRNA stability experiments were performed in Kupffer cells and in RAW264.7 macrophages. Kupffer cells from ethanol- and pair-fed rats were stimulated with 0 or 100 ng/ml of LPS for 60 min. Cells were further incubated in the presence or absence of actinomycin D for 1–2 h. RNA was isolated and TNF-alpha mRNA expression measured by ribonuclease protection assays . Chronic ethanol consumption stabilized LPS-induced TNF-alpha mRNA in Kupffer cells isolated from ethanol-fed rats (t1/2 > 100 min), compared to those isolated from pair-fed rats (t1/2 < 40 min). A similar effect of chronic ethanol was observed on the TNF-alpha mRNA stability in RAW 264.7 macrophages. In control cells, LPS-induced TNF-alpha mRNA decayed with an approximate half-life of 35 min. However, treatment of cells with 25 mM ethanol for 48 h not only increased the accumulation of TNF-alpha mRNA, but also substantially stabilized the TNF-alpha transcript (t1/2 >100 min) . These data demonstrate that exposure to chronic ethanol both in vivo and in vitro results in a marked stabilization of LPS-induced TNF-alpha mRNA.
Inhibition of p38 MAP kinase specifically eliminates ethanol-mediated stabilization of TNF-alpha mRNA
Activation of p38 and ERK1/2 MAP kinases has been linked to mRNA stabilization of otherwise short-lived cytokine and other immediate early response genes [34–39]. We have reported that chronic ethanol exposure, both in vivo and during culture, increases both LPS-stimulated ERK1/2 and p38 activation [19, 20, 30]. Therefore, we asked whether ethanol-induced potentiation of p38 or ERK1/2 MAP kinases was involved in the stabilization of TNF-alpha mRNA observed after chronic ethanol exposure. Kupffer cells isolated from ethanol- and pair-fed rats were pretreated with either 20 micromolar SB203580 or 50 micromolar of PD98059 followed by stimulation with LPS. After stimulation with LPS, Kupffer cells were further treated or not with actinomycin D. Total RNA was isolated and TNF-alpha mRNA levels measured by ribonuclease protection assays . Inhibition of p38 activation completely abrogated ethanol-mediated stabilization of TNF-alpha mRNA . In contrast, inhibition of ERK1/2 activation by PD98059 had no effect on ethanol-mediated stabilization of TNF-alpha mRNA .
Chronic ethanol exposure, either by in vivo feeding to rats or in vitro during macrophage culture, resulted in a stabilization of the LPS stimulated TNF-alpha mRNA transcripts . Interestingly, Motomura and colleagues  recently reported a stabilization of TNF-alpha mRNA transcripts in Kupffer cells isolated from ethanol-fed rats even when they were not stimulated with exogenous LPS. This stabilization was associated with a depletion of retinoic acid during in vivo ethanol exposure . Taken together, these data indicate that chronic ethanol-induced stabilization of TNF-alpha mRNA contributes significantly to increased LPS-stimulated TNF-alpha secretion. Importantly, inhibition of p38 MAPK activity prevented the stabilization of TNF-alpha mRNA and reduced secretion of TNF-alpha in response to LPS . These data suggest that regulation of TNF-alpha mRNA stability mediates increased TNF-alpha production during ethanol consumption and thus contributes to the progression of inflammation during alcoholic liver disease.
- Lieber CS: Alcohol and the liver: 1994 update. Gastroenterology. 1994, 106: 1085-1105.PubMedGoogle Scholar
- Gressner AM, Bachem MG: Molecular mechanisms of liver fibrogenesis – a homage to the role of activated fat-storing cells. Digestion. 1995, 56: 335-346.View ArticlePubMedGoogle Scholar
- Adachi Y, Bradford BU, Gao W, Bojes HK, Thurman RG: Inactivation of Kupffer cells prevents early alcohol-induced liver injury. Hepatology. 1994, 20: 453-460. 10.1016/0270-9139(94)90199-6.View ArticlePubMedGoogle Scholar
- Fukui H, Brauner B, Bode J, Bode C: Plasma endotoxin concentrations in patients with alcoholic and nonalcoholic liver disease: reevaluation with an improved chromogenic assay. J Hepatol. 1991, 12: 162-169. 10.1016/0168-8278(91)90933-3.View ArticlePubMedGoogle Scholar
- Bode C, Kugler V, Bode JC: Endotoxemia in patients with alcoholic and non-alcoholic cirrhosis and in subjects with no evidence of chronic liver disease following acute alcohol excess. J Hepatol. 1987, 4: 8-14. 10.1016/S0168-8278(87)80003-X.View ArticlePubMedGoogle Scholar
- Nanji AA, Khettry U, Sadrzadeh SMH, Yamanaka T: Severity of liver injury in experimental alcoholic liver disease: Correlation with plasma endotoxin, prostaglandin E2, leukotriene B4 and thromboxane B2. Am J Path. 1993, 142: 367-373.PubMed CentralPubMedGoogle Scholar
- Thurman RG: of Hepatic Toxicity II. Alcoholic liver injury involves activation of Kupffer cells by endotoxin. Am J Physiol. 1998, 275: G605-G611.PubMedGoogle Scholar
- Honchel R, Ray M, Marsano L, Cohen D, Lee E, Shedlofsky S, McClain CJ: Tumor necrosis factor in alcohol enhanced endotoxin liver injury. Alcohol Clin Exp Res. 1992, 16: 665-669. 10.1111/j.1530-0277.1992.tb00656.x.View ArticlePubMedGoogle Scholar
- Aldred A, Nagy LE: Ethanol dissociates hormone-stimulated cAMP production from inhibition of TNF-alpha production in rat Kupffer cells. Am J Physiol. 1999, 276: G98-G106.PubMedGoogle Scholar
- Beutler B: TNF, immunity and inflammatory disease: lessons of the past decade. J Investig Med. 1995, 43: 227-235.PubMedGoogle Scholar
- Jacob CO: Tumor necrosis factor alpha in autoimmunity: pretty girl or old witch?. Immunol Today. 1992, 13: 122-125. 10.1016/0167-5699(92)90107-I.View ArticlePubMedGoogle Scholar
- Keffer J, Probert L, Cazlaris H, Georgopoulos S, Kaslaris E, Kioussis D, Kollias G: Transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis. EMBO J. 1991, 10: 4025-4031.PubMed CentralPubMedGoogle Scholar
- Reimund JM, Wittersheim C, Dumont S, Muller CD, Baumann R, Poindron P, Duclos B: Mucosal inflammatory cytokine production by intestinal biopsies in patients with ulcerative colitis and Crohn's disease. J Clin Immunol. 1996, 16: 144-150. 10.1007/BF01540912.View ArticlePubMedGoogle Scholar
- Shalaby MR, Fendly B, Sheehan KC, Schreiber RD, Ammann AJ: Prevention of the graft-versus-host reaction in newborn mice by antibodies to tumor necrosis factor-alpha. Transplantation. 1989, 47: 1057-1061. 10.1097/00007890-198906000-00028.View ArticlePubMedGoogle Scholar
- Tilg H, Diehl AM: Cytokines in alcoholic and nonalcoholic steatohepatitis. New Engl J Med. 2000, 343: 1467-1476. 10.1056/NEJM200011163432007.View ArticlePubMedGoogle Scholar
- McClain CJ, Cohen DA: Increased tumor necrosis factor production by monocytes in alcoholic hepatitis. Hepatology. 1989, 9: 349-351. 10.1002/hep.1840090302.View ArticlePubMedGoogle Scholar
- Khoruts A, Stahnke L, McClain CJ, Logan G, Allen JI: Circulating tumor necrosis factor, interleukin-1 and interleukin-6 concentrations in chronic alcoholic patients. Hepatology. 1991, 13: 267-276. 10.1016/0270-9139(91)92439-F.View ArticlePubMedGoogle Scholar
- Mathurin P, Deng Q-G, Keshavarzian A, Choudhary S, Holmes EW, Tsukamoto H: Exacerbation of alcoholic liver injury by enteral endotoxin in rats. Hepatology. 2000, 32: 1008-1017. 10.1053/jhep.2000.19621.View ArticlePubMedGoogle Scholar
- Kishore R, Hill JR, McMullen MR, Frenkel J, Nagy LE: ERK1/2 and Egr-1 contribute to increased TNF-alpha production in rat Kupffer cells after chronic ethanol feeding. Am J Physiol Gastrointest Liver Physiol. 2002, 282: G6-G15.View ArticlePubMedGoogle Scholar
- Kishore R, McMullen MR, Nagy LE: Stabilization of TNF-alpha mRNA by chronic ethanol: role of A+U rich elements and p38 mitogen activated protein kinase signaling pathway. J Biol Chem. 2001, 276: 41930-41937. 10.1074/jbc.M107181200.View ArticlePubMedGoogle Scholar
- Papadakis KA, Targan SR: Tumor necrosis factor: Biology and therapeutic inhibitors. Gastroenterology. 2000, 119: 1148-1157. 10.1053/gast.2000.18160.View ArticlePubMedGoogle Scholar
- Watkins LR, Hansen MK, Nguyne KT, Lee JE, Maier SF: Dynamic regulation of the proinflammatory cytokine, interleukin-1 beta. Life Sci. 1999, 65: 449-481. 10.1016/S0024-3205(99)00095-8.View ArticlePubMedGoogle Scholar
- Tsai EY, Falvo JV, Tsytsykova AV, Barczak AK, Reimold AM, Glimcher LH, Fenton MJ, Gordon DC, Dunn IF, Goldfeld AE: A lipopolysaccharide-specific enhancer complex involving Ets, Elk-1, Sp1 and CREB binding protein and p300 is recruited to the tumor necrosis factor alpha promoter in vivo. Mol Cell Biol. 2000, 20: 6084-6094. 10.1128/MCB.20.16.6084-6094.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Yao J, Mackman N, Edgington TS, Fan ST: Lipopolysaccharide induction of the tumor necrosis factor alpha promoter in human monocytic cells. Regulation by egr-1, c-jun and NFk beta transcription factors. J Biol Chem. 1997, 272: 17795-17801. 10.1074/jbc.272.28.17795.View ArticlePubMedGoogle Scholar
- Means TK, Pavlovich RP, Roca D, Vermeulen MW, Fenton MJ: Activation of TNF-alpha transcription utilizes distinct MAP kinase pathways in different macrophage populations. J Leukocyte Biol. 2000, 67: 885-893.PubMedGoogle Scholar
- Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, Beutler B: Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science. 1998, 282: 2085-2088. 10.1126/science.282.5396.2085.View ArticlePubMedGoogle Scholar
- Sweet MJ, Hume DA: Endotoxin signal transduction in macrophages. J Leukocyte Biol. 1996, 60: 8-26.PubMedGoogle Scholar
- Guha M, Mackman N: LPS induction of gene expression in human monocytes. Cell Signal. 2001, 13: 85-94. 10.1016/S0898-6568(00)00149-2.View ArticlePubMedGoogle Scholar
- Diamond I, Gordon AS: Cellular and molecular neuroscience of alcoholism. Physiol Rev. 1997, 77: 1-20.PubMedGoogle Scholar
- Shi L, Kishore R, McMullen M, Nagy LE: Chronic ethanol increases LPS-stimulated Egr-1 expression in RAW 264.7 macrophages: contribution to enhanced TNF-alpha production. J Biol Chem. 2002, 277: 14777-147785. 101074/jbc.M108967200PubMedGoogle Scholar
- Lieber CS, DeCarli LM: The feeding of alcohol in liquid diets: two decades of application and 1982 update. Alcohol Clin Exp. 1982, 6: 523-531. 10.1111/j.1530-0277.1982.tb05017.x.View ArticleGoogle Scholar
- Iles K, Nagy LE: Chronic ethanol feeding increases the quantity of G-proteins in rat liver plasma membranes. Hepatology. 1995, 21: 1154-1160. 10.1016/0270-9139(95)90268-6.PubMedGoogle Scholar
- Motomura K, Ohata M, Satre M, Tsukamoto H: Destabilization of TNF-alpha mRNA by retinoic acid in hepatic macrophages: implications for alcoholic liver disease. Am J Physiol Endocrinol Metab. 2001, 281: E420-E429.PubMedGoogle Scholar
- Winzen R, Kracht M, Ritter B, Wilhelm A, Chen CY, Shyu AB, Muller M, Gaestel M, Resch K, Holtmann H: The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism. EMBO J. 1999, 18: 4969-4980. 10.1093/emboj/18.18.4969.PubMed CentralView ArticlePubMedGoogle Scholar
- Holtmann H, Winzen R, Holland P, Eickemeier S, Hoffmann E, Wallach D, Malinin NL, Cooper JA, Resch K, Kracht M: Induction of interleukin-8 synthesis integrates effects on transcription and mRNA degradation from at least three different cytokine- or stress-activated signal transduction pathways. Mol Cell Biol. 1999, 19: 6742-6753.PubMed CentralView ArticlePubMedGoogle Scholar
- Lasa M, Brook M, Saklatvala J, Clark AR: Dexamethasone destabilizes cyclooxygenase 2 mRNA by inhibiting mitogen-activated protein kinase p38. Mol Cell Biol. 2001, 21: 771-780. 10.1128/MCB.21.3.771-780.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Jang BC, Sanchez T, Schaefers HJ, Trifan OC, Liu CH, Creminon C, Huang CK, Hla T: Serum withdrawal-induced post-transcriptional stabilization of cyclooxygenase-2 mRNA in MDA-MB-231 mammary carcinoma cells requires the activity of the p38 stress-activated protein kinase. J Biol Chem. 2000, 275: 39507-39515. 10.1074/jbc.M003224200.View ArticlePubMedGoogle Scholar
- Ridley SH, Dean JL, Sarsfield SJ, Brook M, Clark AR, Saklatvala J: A p38 MAP kinase inhibitor regulates stability of interleukin-1-induced cyclooxygenase-2 mRNA. FEBS Lett. 1998, 439: 75-80. 10.1016/S0014-5793(98)01342-8.View ArticlePubMedGoogle Scholar
- Wang YZ, Zhang P, Rice AB, Bonner JC: Regulation of interleukin-1b-induced platelet-derived growth factor receptor-a expression in rat pulmonary myofibroblasts by p38 mitogen-activated protein kinase. J Biol Chem. 2000, 275: 22550-22557. 10.1074/jbc.M909785199.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.