Role of Hepatocytes in Direct Clearance of Lipopolysaccharide in Rats

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1 GASTROENTEROLOGY 1995;19: Role of Hepatocytes n Drect Clearance of Lpopolysaccharde n Rats YOSHHIRO MIMURA, SHOTARO SAKISAKA, MASARU HARADA, MICHIO SATA, and KYUICHI TANIKAWA Second Department of Medcne, Kurume Unversty School of Medcne, Kururne, Japan Background & Ams: The lver s the clearance organ for lpopolysaccharde (LPS). The am of ths study was to nvestgate the blary excreton of LPS usng fluorescen sothocyanate (FITC)-Iabeled LPS. Methods: After FITC-LPS was njected ntravenously nto rats, the cellular localzaton of fluorescence n the lver was examned and the blary excreton of fluorescence was measured. The effects of gadolnum chlorde, a blocker of Kupffer cells, and colchcne, an nhbtor of mcrotubules, on the blary excreton of fluorescence was nvestgated, and ble was analyzed usng hgh-performance lqud chromatography. Results: Laser scannng confocal mcroscopy showed that fluorescence was taken up by hepatocytes 5 mnutes after njecton of FITC-LPS nto the portal ven. When FITC-LPS was njected nto the portal ven, fluorescence was rapdly secreted nto ble, peakng at 2 mnutes, and 25.1% of the njected dose appeared n ble wthn 6 mnutes. When the same dose of FITC-LPS was njected nto the tal ven, 15.8% appeared n ble wthn 6 mnutes. Chromatography showed that FITC-LPS was excreted nto ble n an unchanged form over a perod of 2 mnutes after njecton. Colchcne sgnfcantly reduced the blary excreton of fluorescence, but gadolnum chlorde had no effect. Conclusons: LPS was drectly and effectvely processed by hepatocytes and secreted nto the ble canalcular system va a mcrotubule-dependent vescular pathway. t ndotoxn s the general term for a class of lpopoly- E t saccharde (LPS) molecules located n the outer cell membrane of gram-negatve enterc bactera. The presence of endotoxn n the systemc crculaton can produce a varety of pathophysologcal effects that are manfested durng gram-negatve nfectons, LPS s therefore consdered a probable cause of septc shock. 1 The lver has been found to play a major role n clearng crculatng LPS from the blood. 2-v In a prevous study n whch 5*Cr-labeled LPS was njected ntravenously nto normal mce, >85% of the dose was found to be trapped n the lver wthn 1 hour.: Hgh concentratons of radoactvty were observed n the gallbladder ble of rabbts gven an ntravenous njecton of 125I-labeled LPS. 8 When *4C-labeled endotoxn was njected nto rats, radoactvty was excreted manly n the feces. 4 The presence of radoactvty n gallbladder ble and feces after ntravenous admnstraton of radolabeled LPS suggests that LPS and/or ts metaboltes may be processed by hepatocytes and secreted nto the blary system. Evdence suggests that LPS s nternalzed, modfed, and released nto the bloodstream by macrophages, ncludng Kupffer cells. 2'5'<8-1. Furthermore, n the lver, a hstochemcal study usng an ant-lps antbody showed that LPS was ntally assocated wth Kupffer cells and later wth hepatocytes. 5 On the other hand, t has been reported that LPS bnds to receptors on hepatocytes n vtro. 12'13 However, the physologcal functon of LPS receptor on hepatocytes has not been well defned. It has not been shown whether hepatocytes take up LPS wthout pror metabolsm by Kupffer cells and secrete t nto ble, although t s known that a pecular structure of LPS was not metabolzed and secreted nto ble by hepatocytes. 14 To show the role of hepatocytes n LPS clearance, we nvestgated the cellular dstrbuton and blary excreton of fluorescen sothocyanate (FITC)-labeled Eschercha col O55:B5 LPS n rats. In addton, we studed the effects of gadolnum chlorde (GdC13), a blocker of Kupffer cells, and colchcne, an nhbtor of mcrotubules, on the blary excreton of LPS. Materals Chemcals and Methods FITC-labeled and -unlabeled LPS were purchased from Lst Bologcal Laboratores Inc. (Campbell, CA). LPS was prepared from E, col (serotype O55:B5) usng phenol extracton and purfed chromatographcally. FITC-LPS has 6.4 mol of FITC (somer I) per mole of-lps (techncal data of FITC-LPS). Abbrevatons used n ths paper: FITC, fluorescen sothocyanate; GdCla, gadolnum chlorde; HPLC, hgh-performance lqud chromatography; LPS, lpopolysaccharde by the Amercan Gastroenterologcal Assocaton /95/$3.

2 197 MIMURA ET AL. GASTROENTEROLOGY Vol. 19, No. 6 The same lots were used for one set of experments. The two LPS preparatons were stored at 4 C and dssolved n sotonc, sterle salne before use. FITC-labeled bovne albumn (FITCalbumn), colchcne, and lumcolchcne were purchased from Sgma Chemcal Co. (St. Lous, MO). FITC somer and sodum pentobarbtal were obtaned from Polyscences Inc. (Warrngton, PA) and Abbott Laboratores (North Chcago, IL), respectvely. The acetontrle used n the moble phase was of hgh-performance lqud chromatography (HPLC) grade, and all other chemcals were of analytcal-reagent grade. Laser Scannng Confocal Mcroscopy The cellular dstrbuton of FITC-LPS n the lver was examned n normal and GdC13-treated rats. FITC-LPS was njected nto the portal ven at a dose of 5 btg/3 g body wt. Lvers were then perfused n stu wth 5 ml ofperodatelysne-paraformaldehyde medum to remove gross blood and for fxaton 5, 1, 2, or 6 mnutes after njecton of FITC- LPS. Furthermore, peces of the lver tssue were fxed n 4% phosphate-buffered formaldehyde for 12 hours and processed for paraffn embeddng. Lver sectons (5 btm) were examned usng laser scannng confocal mcroscopy (LSM-GB2; Olympus, Tokyo, Japan) at an exctaton wavelength of 488 nm and an emsson wavelength of 515 nm. Expermental Protocol Male Wstar rats weghng g and bred under specfc pathogen-free condtons were used n all experments, whch were performed between 1 AM and 4 PM. After rats were anesthetzed wth ntrapertoneal njecton of sodum pentobarbtal (Nembutal, 39 mg/kg body wt), the common ble duct was exposed and cannulated wth a polyethylene catheter, whch had outer and nner dameters of 9 and 32 ~tm, respectvely, and a length of 1 cm. Ble collecton for experments was started 3-45 mnutes after ble duct cannulaton when ble flow became stablzed. Usng a 27-gauge needle, we njected 5 p.g/3 g body wt of FITC-LPS or FITC-albumn n.5-ml of salne nto a branch of the portal ven or the tal ven. Control rats were gven the same amount of unlabeled LPS n.5 ml of normal salne. Ble was collected every 1 mnutes for 7 mnutes after admnstraton of FITC- LPS whle the abdomen was covered wth wet gauze. The collected ble was centrfuged at 5 rpm for 15 mnutes at room temperature. The concentraton of the FITC moety n the supernatant was determned usng an F3 fluorescence spectrophotometer (Htach Ltd., Tokyo, Japan) at an exctaton wavelength of 496 nm and an emsson wavelength of 517 nm. In a prelmnary study, the fluorescent ntensty of FITC-LPS dssolved n ble was lnearly related to FITC-LPS concentraton rangng from.1 to 1 ~g/ml. Thus, the FITC- LPS concentraton n ble was determned by ths standard curve. The FITC moety represents the amount of unchanged LPS and/or a metabolc product carryng the FITC molecule. GdC13, a blocker of Kupffer cells, was admnstered ntravenously (5 mg/kg body wt) 24 hours before admnstraton of FITC-LPS to assess the partcpaton of Kupffer cels n clearance of LPS. Colchcne, an nhbtor of mcrotubules, and lumcolchclue, an somer of colchcne, were admnstered ntrapertoneally (1 gg/g body wt) 4 hours before the admnstraton of FITC-LPS to nvestgate the ntercellular transport and processng knetcs of FITC-LPS n the lver. In addton, we examned the relatonshp between the njected dose of FITC-LPS at a concentraton of 5-6 /.tg/3 g body wt and an amount of the FITC moety secreted nto ble. HPLC of FITC-LPS To determne whether the LPS secreted n ble was unchanged or represented a metabolc product carryng the FITC molecule, we analyzed ble usng HPLC accordng to the method of Martn and Ghabral. 15 Ble obtaned over a perod of 2 mnutes after admnstraton of FITC-LPS was centrfuged at 5 rpm for 15 mnutes at room temperature. The supernatant was collected and then dssolved n 2 volumes of chloroform-methanol (1:2 by volume). The lower phase contents were dred, and the resdue was redssolved n 2 ml of dstlled water and centrfuged at 1, rpm for 3 mnutes at 4 C. Chromatography was performed on 2 btl of the supernatant usng a constant-flow hgh-pressure lqud chromatograph consstng of an LC-6A pump system equpped wth an SCL-6A controller and an RF-53 spectrofluorometer (Shmadzu, Kyoto, Japan) at an exctaton wavelength of 496 nm and an emsson wavelength of 517 nm wth exctaton and emsson slt wdths of 1 nm. The column used was an Ultrahydrogel lnear column (ID, mm; Waters Assocates, Mlford, MA) packed wth a gel of cross-lnked hydroxylated polymethacrylate. The moble phase was 1 mmol/l phosphate buffer (ph 7.75) contanng 2% (vol/vol) acetontrle at a flow rate of.4 ml/mn. Statstcal Analyss Statstcal analyss of the data was performed usng Student's t test. P values of <.5 were consdered to ndcate sgnfcance of dfferences between means. Results Cellular Dstrbuton of FITC-LPS n the Lver Laser scannng confocal mcroscopy revealed fluorescence n hepatc macrophages (Kupffer cells) and hepatocytes of lver specmens 1 mnutes after 5 ~g/3 g body wt of FITC-LPS was njected nto a branch of the portal ven (Fgure 1A). Fluorescence ntensty was stronger n Kupffer cells than n hepatocytes. Fant but sgnfcant fluorescence was detected n Kupffer cells and hepatocytes 5 mnutes after njecton (data not shown). When FITC alone was njected, cellular fluorescence was much weaker, and ts ntensty was smlar n Kupffer cells and hepatocytes

3 December 1995 HEPATOCYTIC CLEARANCE OF LIPOPOLYSACCHARIDE 1971 Blary Excreton of FITC-LPS Nether 5 ~g/3 g body wt of FITC-LPS (Fgure 2A) nor the same dose of unlabeled LPS (data not shown) affected ble flow durng a 7-mnute perod after the portal ven admnstraton. Excreton of the FITC moety nto ble peaked at 2 mnutes, gradually decreasng durng the next 5 mnutes (Fgure 2B). The cumulatve amount of the FITC moety excreted nto ble was 25.1% + 5.7% (mean SD; n = l l ) of the njected dose of FITC-LPS at 6 mnutes (Fgure 2C). Blary Excreton of FITC-LPS at the Varous Injected Doses When 5-6 ~g/3 g body wt of FITC-LPS was njected nto the portal ven, the blary excreton of the FITC moety ncreased gradually accordng to the njected dose of FITC-LPS (Fgure 3A). The peak concentraton of the excreted FITC moety grew n a lnear fashon (Fgure 3B). However, admnstraton of >8 btg/3 g body wt of FITC-LPS reduced ble flow (data not shown). Blary Excreton of FITC-Albumn Fgure 1. Laser scannng confocal mcrograph of fluorescence n the rat lver. (A) Ten mnutes after portal ven njecton of 5 pg/3 g body wt of FITC-LPS. Fluorescence was localzed n hepatocytes and Kupffer cells (arrows) n the snusodal space. (B) Sxty mnutes after portal ven njecton of FITC alone. Fluorescence ntensty was not dfferent between hepatocytes and Kupffer cells when FIT(:; alone was njected. (C) Sxty mnutes after portal ven njecton of FITC-LPS. Fluorescence was detected n hepatocytes and nonparenchymal cells, and fluorescence ntensty was reduced along the acnus of the lver. Kupffer cells (arrows) showed stronger fluorescence than hepatocytes. C, central ven; P, portal area. (Fgure 1B). Sxty mnutes after njecton of FITC-LPS, fluorescence was observed manly n hepatocytes and nonparenchymal cels close to the termnal portal venule, and fluorescence ntensty was reduced n the percentral area (Fgure 1C). Fluorescence was observed wthn the hepatc acnus from 5 to 6 mnutes after njecton of FITC-LPS. Nether margnaton of neutrophls and mononuclear cells nor fbrnous deposts n the hepatc snusods and the spaces of Dsse were observed wthn 6 mnutes after njecton of FITC-LPS (Fgure 1C). Furthermore, the hepatc acnus was composed of normal-appearng hepatocytes (Fgure 1C). The fluorescence for FITC-LPS was also dstrbuted n splenc macrophages and pulmonary dust cels (data not shown). When 5 J,tg/3 g body wt of FITC-albumn was njected nto the portal ven, excreton of the FITC moety dd not peak at 2 mnutes (Fgure 4A) and only 2% of the njected dose appeared n ble wthn 3 mnutes (Fgure 4B). HPLC of FITC-LPS Chromatograms showed that fluorescent peaks of the prenjected FITC-LPS (Fgure 5A) and the FITC moety secreted nto ble (Fgure 5B) had the same retenton tme. Blary Excreton After Portal Ven and Systemc Injectons of FITC-LPS Blary excreton of the FITC moety by the lver after ntravenous njecton va the tal ven was sgnfcantly lower than that after portal ven njecton va the mesenterc ven (Fgure 6). Sxty mnutes after ntravenous admnstraton of FITC-LPS, 15.8% + 4.7% (mean SD; n = 5) of the njected dose appeared n ble. Blary excreton of the FITC moety n rats gven a systemc njecton wth FITC-LPS decreased to 62.9% of that found n rats gven a portal ven njecton (P <.1). Effect of GdCla on Blary Excreton of FITC-LPS When 5 btg/3 g body wt of FITC-LPS was njected ntraportally nto GdC13-treated rats, blary ex-

4 1972 MIMURA ET AL. GASTROENTEROLOGY Vol. 19, No. 6 creton of the FITC moety was the same as n control anmals (Fgure 7). Uptake of FITC-LPS by Kupffer Cells Pretreated Wth GdCla The fluorescence ntensty n Kupffer cells was reduced by GdCI 3 1 mnutes after njecton of FITC-LPS (Fgure 8). GdC13 blocked endocytoss of FITC-LPS by Kupffer cells from 5 to 6 mnutes after njecton of FITC-LPS. Effect of Colchcne on Blary Excreton of FITC-LPS Sxty mnutes after admnstraton of FITC-LPS, 17.8% + 2.7% (mean + SD; n = 8) of the njected A 25 2 lso O m I 5 a 2' p V1mn II[- I I I I I I I dose appeared n ble when 5 [.tg/3 g body wt of FITC-LPS was njected ntraportally nto colchcnetreated rats. Colchcne, an nhbtor of mcrotubules, sgnfcantly nhbted blary excreton of the FITC moety to 7.9% of control wthn 6 mnutes after admnstraton of FITC-LPS (Fgure 9). On the other hand, lumcolchcne, an somer of colchcne that does not affect mcrotubules, dd not affect blary excreton of the FITC moety (data not shown). Colchcne and lumcolchcne dd not affect ble flow. Dscusson The lver s the man clearance organ for LPS, and Kupffer cells have been reported to be responsble for hepatc clearance of LPS. 2'5'6's However, t s not well known whether the parenchymal lver cells (hepatocytes) are responsble for the drect clearance of LPS, whether hepatocytes take up LPS and secrete t nto ble, or whch transport system excretes LPS nto ble from the lver. In the present study, we studed the route and rate of n vvo excreton of LPS n normal rats usng FITC-LPS. The FITC molecule n FITC-LPS has been found to bnd covalently to amne groups of LPS. 15 FITC-LPS was postve for the lmulus amebocyte lysate assay (our unpublshed observaton). FITC-LPS s complexed to LPS-bndng proten, and the resultng complex s recognzed by CD14 receptor on monocytes n vtro) 6 FITC-LPS also g.~ "" 1' A t,~nl B,,g/~ 4- C 3' % of njected FITC-LPS,~ 3! /,, N 2,.//,g (r=.82217)., //,,, / '* ~ 1'., / // 1# Mnutes after njecton Fgure 2. Blary excreton of FITC moety after portal ven njecton of 5 #g/3 g body wt of FITC-LPS. (A) Effect of FITC-LPS on ble flow. (B) Excreton of FITC moety nto ble quantfed as fluorescence ntensty. (C) Cumulatve amount of the FITC moety excreted nto ble. Results are expressed as mean _+ SD obtaned from 11 rats. 2 4O 6O 8 Mtres after njecton /~*' e J,,, - ) Injected dose ( b~ g/3 gm body wt) Fgure 3. Relatonshp between the njected dose of FITC-LPS and blary excreton of the FITC moety. (A) Representatve data of blary excretons of the FITC moety after portal ven njecton of 5-6 #g/ 3 g body wt of FITC-LPS. (B) The peak concentraton of the secreted FITC moety n A s plotted along the ordnate aganst the njected dose of FITC-LPS on the abscssa.

5 December 1995 HEPATOCYTIC CLEARANCE OF BPOPOLYSACCHARIDE 1973 actvates B cells n vvoj 7'*s In our confocal mcroscopy study, fluorescence of FITC-LPS was predomnantly dstrbuted n splenc macrophages and pulmonary dust cells as well as n Kupffer cells after venous njecton of FITC- LPS. Ths cellular dstrbuton was smlar to that of LPS found n prevous studes usng autoradography or mmunohstochemcal methods. ~'5'<8 These results suggested that FITC-LPS could be used as a tracer of LPS. Electron-mcroscopc autoradography, cell solaton, and partal purfcaton of cell types showed that 15Crlabeled LPS was taken up by Kupffer cells, endothelal cells, and hepatocytes n rats..9 Van Bossuyt et al. showed that 3H-labeled LPS was localzed n the mtochondra, cell membranes, and the perphery of hepatocytes 15 mnutes after njecton of the tracer. 2 In the present study, laser scannng confocal mcroscopy also showed hepatocytc uptake of FITC-LPS. Mcroscopy showed that FITC-LPS was processed by hepatocytes as well as by Kupffer cells 5 mnutes after njecton va the mesenterc ven. Uptake was greater n hepatocytes of acnar zones 1 and 2 than n those of acnar zone 3, probably because hepatocytes surroundng the termnal portal re- nule (acnar zone 1) were exposed to a hgh concentraton of FITC-LPS. In the present study, blary excreton of the FITC moety ncreased rapdly, peakng 2 mnutes after njecton of FITC-LPS, unlke FITC-albumn. Colchcne nhbted the blary excreton of FITC-LPS. The blary secretory curve of FITC-LPS was smlar to those found n prevous studes for mmunoglobuln A, 21 horseradsh peroxdase, 22'23 nsuln, 24 nuln, 25 and copper, 26 whch appeared to be transported drectly to ble wthout passng through the lysosomal compartment of hepatocytes. Because colchcne was known to nterfere wth mcrotubules and related membrane functons, 22'25-27 these resuts suggested that FITC-LPS was transported to ble va a mcrotubule-dependent vescular pathway that rapdly transverses hepatocytes from the snusodal membrane to the ble canalculus. LPS has been found to be taken up n all organs by A -r., CO ~t 3 p g/ml ~ 2' g F1TC-LPS ~ 1 F1TC-AIb! B ~ ~.... % of njected dose 2O!1o ~ FITC-LPS aj oj Mnutes after njecton FITC-Alb Fgure 4. Blary excreton of the FtTC moety after portal ven njecton of 5 pg/3 g body wt of FITC-albumn (Alb) (O) and FITC-LPS (). (A) Excreton of FITC moety nto ble quantfed as fluorescence ntensty. (B) Cumulatve amount of the FITC moety excreted nto ble. Results of FITC-albumn admnstraton are expressed as mean ± SD obtaned from 3 rats. 4 B. I I Retenton tme 4 ran Fgure 5, HPLC of (A) prenjected FITC-LPS and (B) FITC moety n collected ble run on an Ultrahydrogel lnear column at a flow rate of.4 ml/mn.

6 1974 MIMURA ET AL. GASTROENTEROLOGY Vol. 19, No. 6 granulocytes and macrophages, ncludng Kupffer cells, and then LPS or ts metaboltes are later redstrbuted nto hepatocytes. 28'29 However, t probably takes longer than 2 mnutes for phagocytc cells to process LPS; then LPS and/or ts metaboltes are transferred from the blood stream to ble through hepatocytes. In the present study, we used 5 [.tg/3 g body wt of FITC-LPS. It s possble that the hgh dose of LPS saturates the Kupffer cell pathway, resultng n preferental processng by hepatocytes. However, fluorescence ntensty n Kupffer cells after njecton of 5 /.tg/3 g body wt of FITC-LPS was weaker than after an njecton of 7 g/3 g body wt (our unpublshed observaton), suggestng that the Kupffer cell was unsaturated wth LPS after njecton of 5 btg/3 g body wt of FITC- LPS. Furthermore, our dose-excreton data showed that the blary efflux of FITC moety ncreased wth the hgh dose of njected FITC-LPS, representng a straght lne passed through the nearby orgn of the coordnates (Fgure 3B). Ths suggested that hepatocytes may partcpate o A. t* glomn n LPS clearance at the low concentraton of LPS n the blood. These fndngs suggested that hepatocytes and Kupffer cells ndependently took up LPS from the blood stream. Admnstraton of 5 /.tg/3 g body wt of FITC- LPS had no effect on ether the rate of ble flow or the hstologcal alteratons n varous tssues. However, admnstraton of >8 ~g/3 g body wt of FITC-LPS reduced ble flow so that we could not examne the maxmum capacty of hepatocytes n blary secreton of LPS. After ntravenous njecton of radolabeled LPS, the radoactvty s secreted nto the blary system. 4'8 After njecton of unlabeled LPS, ~-hydroxymyrstc acd, a pecular structure of lpd A moety n LPS, s also secreted nto ble. 14 However, t s not known whether the molecules secreted nto ble were ntact LPS or ts metaboltes. Our HPLC suggested that FITC-LPS was processed and secreted nto ble by hepatocytes predomnantly n an unmetabolzed form durng a perod of 2 mnutes after njecton. Intravenous njecton of GdC13 strongly reduced lver uptake of collodal carbon and radoactvely labeled for g 2. B 3 ] % of njected FITC-LPS 1. m. B 3O ~8 m -! m e m "~ 1' Mnutes after njecton 1' m u 4 5! 6 m 7 8 Mnutes after njecton Fgure 6. Blary excreton of the FITC moety after portal ven and systemc njectons of FITC-LPS. Ffty mcrograms per 3 g body wt of FITC-LPS was admnstered va the mesenterc ven () or the tal ven (t). (A) Blary output of the FITC moety. (B) Cumulatve amount of the FITC moety n ble. Results of systemc njectons of FITC-LPS are expressed as mean -- SD obtaned from 3 rats. **P <.5; ***P <.1. Fgure 7. Effect of GdCI3 on blary excreton of the FITC moety after portal ven njecton of FITC-LPS, Ffty mcrograms per 3 g body wt of FITC-LPS was admnstered nto GdCl3-treated rats ( ) and control rats (e), (A) Blary output of the FITC moety. (B) Cumulatve amount of the FITC moety n ble. Results n GdCls-treated rats are expressed as mean ± SD obtaned from 6 rats, There was no statstcal sgnfcance between the groups.

7 December 1995 HEPATOCYTIC CLEARANCE OF LIPOPOLYSACCHARIDE 1975 egn erythrocytes) It s lkely that accumulaton of these partcles by the lver s manly caused by phagocytoss by Kupffer cells, j The present study also showed that GdC13 nhbted endocytoss of FITC-LPS by Kupffer cells. However, GdCI 3 dd not reduce the blary secreton of the FITC moety by hepatocytes. These fndngs suggest that Kupffer cells may not partcpate n the early excreton of LPS nto ble. If all of the FITC-LPS had bound to some plasma protens or been processed by cels other than hepatocytes, there would probably have been no dfference n the blary excreton of the FITC moety between perpheral and portal ven admnstraton. However, hepatc clearance of the FITC moety n rats gven systemc njectons decreased to 62.9% of that n anmals gven portal ven njectons. Portal ven admnstraton of FITC-LPS would ncrease the concentraton of LPS delvered to hepatocytes compared wth systemc admnstraton. Our results suggest that hepatocytes may take up FITC-LPS drectly and excrete t nto ble. Prevous studes have shown that LPS admnstered va the portal ven s cleared more effectvely than that admnstered by other routes, 31'32 whch may explan why portal ven njecton of LPS was assocated wth a lower mortalty rate than systemc admnstraton n rats. 33 Recently, Parent showed that rat hepatocytes had membrane receptors for LPS. 1~ However, the physologcal functon of hepatocyte LPS receptors s unclear. The receptors may be related to the drect clearance of LPS to ble. In ths study, a sgnfcant amount of LPS was drectly and effectvely removed from the crculaton and trans- Fgure 8. Laser scannng confocal mcrograph of fluorescence n the lver pretreated wth GdCI3. Ten mnutes after portal ven njecton of 5 btg/3 g body wt of FITC-LPS. Compare wth Fgure 1A. GdCl3 blocked endocytoss of FITC-LPS by Kupffer cells. O2!'1 O 6 8 2" 1" ol 3-2 1' ' % of njected FITC-LPS T l ~t~~4~ ~ Mnutes after njecton Fgure 9. Effect of colchcne on (A) blary output and (B) cumulaton amount of the FITC moety secreted nto ble after portal ven njecton of 5 btg/3 g body wt of FITC-LPS. e, Colchcne-pretreated rats; O, data from control rats as n Fgure 2. Results n colchcne-pretreated rats are expressed as mean _+ SD obtaned from 8 rats. **P <.5; ***P <.1. ferred to ble by lver parenchymal cells through a mcrotubule-dependent vescular pathway. The rest of the njected LPS mght be cleared by phagocytc cells (granulocytes, monocytes, and macrophages), as descrbed prevously. 2,5,6,8 Studes have shown that endotoxema s assocated wth lver dseases such as obstructve jaundce, 34 cholestass, ~5 and lver crrhoss) 6 Dsturbed blary excreton s also assocated wth these dseases, 3v suggestng that a dsturbance n LPS excreton n addton to ncreased absorpton of LPS from the ntestne 38 may contrbute to endotoxema. References 1. Nogare ARD. Southwestern Internal Medcne Conference: septc shock. Am J Med Sc 1991;32: Carey F J, Braude AL, Zalesky M. Studes wth radoactve endotoxn. III. The effect of tolerance on the dstrbuton of radoactvty after ntravenous njecton of Eschercha col endotoxn labelled wth 51Cr. J Cln Invest 1958;37: Braude AI, Carey FJ. Zalesky M. Studes wth radoactve endotoxn. II. Correlaton of physologcal effects wth dstrbuton of radoactvty n rabbts njected wth lethal doses of E. col endo-

8 1976 MIMURA ET AL. GASTROENTEROLOGY Vol. 19, No. 6 toxn labelled wth radoactve sodum chromate. J Cln Invest 1955; 34: Klen B, Freudenberg MA, Galanos C. Excreton of radoactvty n faeces and urne of rats njected wth 3H,14C-lpopolysaccharde. Br J Exp Pathol 1985;66: Freudenberg MA, Freudenberg N, Galanos C. Tme course of cellular dstrbuton of endotoxn n lver, lungs and kdneys of rats. Br J Exp Pathol 1982;63: Musson RA, Morrson DC, Uevtch RJ. Dstrbuton of endotoxn (lpopolysaccharde) n the tssues of lpopoysaccharde-responsve and unresponsve mce. Infect Immun 1978;21: Zlydaszyk JC, Moon RJ. Fate of 5~Cr-labeled lpopolysaccharde n tssue culture cells and lvers of normal mce. Infect Immun 1976; 14: Mathson JC, Ulevtch RJ. The clearance, tssue dstrbuton and cellular localzaton of ntravenously njected lpopolysacchadde n rabbts. J Immunol 1979; 123: Peterson AA, Munford RS. Dephosphorylaton of the lpd A moety of Eschercha col lpopolysaccharde by mouse macrophages. 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Scand J Gastroenterology Suppl 1992; 194: Tachyama G, Sakou M, Kambayash J, ljma S, Tsujnaka T, Mor T. Endogenous endotoxema n patents wth lver crrhoss--a quanttatve analyss of endotoxn n portal and perpheral blood. Jpn J Surg 1988;18: Klaassen CD, Watkns III JB. Mechansms of ble fo;maton, hepatc uptake, and blary excreton. Pharmacol Rev 1984;36: Kocsar LT, Bertok L, Varteresz V. Effect of ble acds on the ntestnal absorpton of endotoxn n rats. J Bacterol 1969;1: Receved February 27, Accepted July 28, Address requests for reprnts to: Yoshhro Mmura, M.D., Second Department of Medcne, Kurume Unversty School of Medcne, 67 Asah-mach, Kurume 83, Japan. The authors thank T. Yasukouch, T. Shnozak and Y. Ogou for expert techncal assstance and Drs. H. Yoshda, K. Noguch, S. Shakado, and M. Yoshtake for dscusson of concepts n ths manuscrpt.