INFECTION AND IMMUNITY, Aug. 1975, p. 411-418Copyright i 1975 American Society for Microbiology

Vol. 12, No. 2Printed in U.S.A.

In Situ Separation of Bacterial Trapping and Killing Functionsof the Perfused Liverl

ROBERT J. MOON,* RUTH A. VRABLE, AND JOYCE A. BROKA

Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824

Received for publication 10 April 1975

CF-1 mice cleared and killed 80% of a 1.2 x 10' intravenous dose of Salmonellatyphimurium after 30 min. The perfused mouse liver trapped 70% of a similardose of S. typhimurium in a single pass, but in the perfusion model no significantkilling of the trapped organisms was observed. The perfused rat liver also avidlytrapped bacteria. Because of its larger size, we have been able to devise tech-niques to experimentally distinguish between the bacterial trapping and killingfunctions of this organ. When the liver was washed free of blood with sterileM-199, over 70 to 80% of a 106 to 1010 dose of viable S. typhimurium was trappedafter a single pass, but no significant bacterial killing was observed. When bloodor plasma was added to the perfusion medium, over 50% of the trapped bacteriawere killed in 15 to 30 min. Phase contrast and electron micrographs of perfusedlivers showed extensive extracellular trapping of bacteria in the sinusoids. Ourdata show that humoral factors are apparently not necessary for efficient trap-ping of live Salmonella by the perfused rat liver but are an absolute requirementfor bactericidal activity of the organ.

The isolated perfused liver has been usedperiodically to study bacterial phagocytosis bythe liver. As early as 1916 Manwaring and Coe(15) and Manwaring and Fritschen (16) estab-lished "laws" of tissue affinity and providedimportant insights into opsonins of immuneserum. These workers, using dogs as their exper-imental animal, perfused a variety of organsand showed that only spleen and liver demon-strated significant affinity for bacteria. By 1930these studies and others (1) had established theimportance of the reticuloendothelial system inhost defense against bacterial invasion. Littlesignificant progress in using perfused liver inhost-parasite interactions was made until 1958,when Howard and Wardlaw (6,7) used in situperfused rat liver to compare phagocytic ratesin different suspending media. Their resultsindicated that normal serum exerted an opsoniceffect for a number of different bacterial strainsand they concluded that antibody, comple-ment, and another heat-labile factor all hadactivity. More recently Bonventre (4) and Jeu-net et al. (8-11) have used the perfused liver tostudy selected aspects of phagocytosis, intracel-lular killing, and blockade of the Kupffer cellsystem. Clearance of Candida albicans by theperfused rat liver has also been recently de-scribed (3).

1 Journal article no. 7185 from the Michigan AgriculturalExperiment Station.

There have been significant advances in re-cent years in understanding the molecular in-teractions of opsonins (19) and polymorphonu-clear leukocytes (22-24). Such studies have ledus to reinvestigate the possible use of theperfused liver technique to study and hopefullyincrease understanding of the functional proc-esses at work in the macrophage system of theliver, as well as expand our understanding of therole of the liver Kupffer cell in defending thehost against bacterial invasion. We report herethe development of an in vitro system whichapproximates the in vivo clearance of intrave-nously injected viable bacteria from the blood.With this system we have been able to experi-mentally distinguish between bacterial trap-ping and bactericidal functions of the liver.

MATERIALS AND METHODSAnimals. Carworth Farms (CF-1) female mice, 18

to 22 g, were purchased from Carworth, Inc., Portage,Mich. Sprague-Dawley male rats, 300 to 400 g, wereobtained from Spartan Research Animals Inc. (Has-lett, Mich.). All animals were maintained understandard laboratory conditions. Purina LaboratoryChow and water were available ad libitum.

Bacteria. Salmonella typhimurium, strain SR-11,was used in all experiments. Overnight cultures of theorganism, grown in brain heart infusion broth, werecentrifuged at 8,000 rpm for 15 min. Cells werewashed once, suspended in saline, and diluted insaline to appropriate concentration. Pour plates of

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412 MOON, VRABLE, AND BROKA

appropriately diluted samples were used for quantita-tion.

Liver perfusion: mouse surgery. Procedures formouse liver perfusion have recently been described(2). Immediately before surgery mice were injected in-travenously with 200 U of sodium heparin (UpjohnCo., Kalamazoo, Mich.) followed by intravenous injec-tion of 1.5 mg of pentobarbital sodium injection (HaverLockhart Laboratories, Shawnee, Kan.). A long ab-dominal incision was made and the intestine was ma-nipulated to expose both the portal vein and the in-ferior vena cava. Two nylon sutures were inserted un-der the portal vein and one was inserted under thevena cava above the right renal vein. Cannulas madefrom 20-gauge needles were filed until the beveled endwas blunt and the shaft roughened. All cannulas wereautoclaved before use.The portal vein was held taut with forceps about 1

cm from the liver, and a small incision was made inthe vein between the forceps and the liver. A cannulafilled with sterile M-199 was inserted in the vein andtied in place with the two sutures. When the cannulawas in place it was attached to Tygon tubing (innerdiameter [lD], 0.13 inch; outer diameter [OD], 0.16inch) which led to a three-way valve (Becton-Dickin-son & Co., model no. MS3033), one port of which helda 1-ml syringe containing sterile M-199 and thesecond port of which was attached to two 50-ml glasssyringes filled with prewarmed, sterile M-199. Afterattachment to the reservoir, the perfusion wasstarted. To avoid swelling of the liver, the inferiorvena cava was bisected below the right kidney to al-low the perfusion medium to flow in through the por-tal vein and out the inferior vena cava.The thoracic cavity was then opened and a ligature

was placed under the superior vena cava above theliver. A small incision was made in the right atrium,and the efferent metal cannula was inserted, tied inplace, and attached to a 15- to 20-cm piece of Tygontubing (ID, 0.31 inch, OD, 0.93 inch).

Immediately after the second cannula was in place,the ligature above the right renal vein was tied,diverting the flow of M-199 into the efferent cannula.The time from tying the ligatures on the portal vein tocompletion of the procedure did not exceed 5 min.The liver was kept moist by bathing it with sterileM-199 and covering with a glass petri dish to slowevaporation.Rat surgery. Rats were anesthetized by intraven-

ous injection of 15 mg of pentobarbital sodium injec-tion (Haver-Lockhart Laboratories, Shawnee, Kan.)into the dorsal vein of the penis. This was followed im-mediately by intravenous injection of 2,000 U of so-dium heparin (Upjohn Co., Kalamazoo, Mich.) dilutedin saline. At this time rats were sometimes bled bycardiac puncture.A full-length medial incision was made and the

ventral walls of the abdomen were retracted. Thestomach and intestine were moved aside to expose theportal vein. Two ligatures were inserted, approxi-mately 1 mm apart, under the portal vein between theliver and the splenic vein. A third ligature was placedunder the inferior vena cava above the right renalvein. The portal vein was held taut by a pair of forceps

1 inch (about 2.5 cm) below the ligatures, and a smallincision was made in the vein. A sterile polyethylenecannula (Becton-Dickinson Co., Rutherford, N.J.: ID,0.046 inch; OD, 0.066 inch) was inserted into the veinand tied in place by both ligatures. The cannula led toa reservoir of M-199 identical to that described above.The valve leading to the reservoir was opened to fillthe cannula with M-199 and to prevent any air fromentering the portal vein. It was immediately closedafter insertion to prevent swelling of the liver. Theposition of the cannula tip was adjusted so that it didnot project beyond the main trunk of the vein into thehepatic tree. After opening the thorax a ligature wasinserted under the inferior vena cava, and the efferentpolyethylene cannula (ID, 0.046 inch; OD, 0.066 inch)was inserted through an incision in the wall of theright atrium, pushed down the inferior vena cava to apoint just above the liver, and tied. At this time thesuture around the inferior vena cava above the rightrenal vein was also tied and the perfusion wasresumed. The time between tying the ligatures on theportal vein and completion of the procedure did notexceed 5 min. The liver was kept moist by bathingwith sterile M-199 and covered with a glass petri dishto slow evaporation.

After surgery, the procedures for mouse and ratliver perfusion were essentially identical. The can-nulated livers were washed with M-199 until theeffluent was clear of blood cells. Careful adjustment ofthe flow rate was necessary during the first fewminutes of the perfusion until equilibrium of thecirculation through the liver had been established andconstant flow could be maintained without difficulty.The effluent was tested for sterility, and any livereffluent having greater than 10 colony-forming units(CFU) of bacteria per ml at this point was omittedfrom the final tabulation of data.

After washing, the 1-ml syringe on the three-wayvalve which contained M-199 was replaced with asyringe filled with 1 ml of S. typhimurium. Thebacteria were slowly and steadily infused and followedimmediately by 100 ml of M-199 for rats or 50 ml ofM-199 for mice. The effluent was collected from theefferent cannula into a sterile 100-ml graduatedcylinder. An additional 1-ml portion was obtainedafter perfusion was completed. This portion wasplated, and the data consistently show that less than0.001% of the infused bacteria continued to be washedout of the liver. Perfusions in this study lasted 30 minor less and were carried out at room temperature. Nospecific attempts to maintain liver oxygenation weremade.The effluent was kept in an ice bath until quantita-

tive tryptose agar pour plates were made. The percentof nontrapped bacteria was calculated by the formula:

number of CFU recovered in effluentnumber ofCFU infused

x 100

The difference between the percentage of recovery inthe effluent and the total infused (100%) suggestedthe percentage of bacteria trapped by the liver. Theliver was disconnected from the perfusion apparatusand, where indicated, homogenized in 100 ml of sterile

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ANTIBACTERIAL FUNCTIONS OF PERFUSED LIVERS 413

saline in a Waring blender for rats or 10 ml of sterilesaline in a glass Teflon homogenizer for mice. Quanti-tative tryptose agar pour plates were made from thehomogenate to determine the number of trappedbacteria which remained viable in the liver. Thepercentage of viable bacteria trapped in the liver plusthe percentage of recovery in the effluent wash couldthen be subtracted from the percentage of bacteriainfused (100%) to give the percentage of killing.Phase and electron microscopy. Liver sections

were fixed for electron microscopy according to themethods of Sabatini et al. (20) with only minormodifications. Ten (rat) or 3 (mice) ml of 3.0%glutaraldehyde in 0.1 M phosphate buffer (pH 7.2)was infused directly into the portal vein. Approxi-mately 25 1-mm3 sections of the right ventral lobewere placed in cold glutaraldehyde overnight. Afterfixation, the liver slices were rinsed three times incold buffer and then refixed in 1.0% osmium tetroxidedissolved in 0.2 M S-collidine buffer (pH 7.4) at 4 Cfor 2 h. Dehydration of all fixed tissues was carried outin increasing concentrations of ethanol. The livertissue was embedded in Epon (14) and sliced on an

LKB Ultratome III. Electron microscope observationswere made on a Philips EM-300, and phase micro-graphs were prepared using a Zeiss phase contrastmicroscope equipped with a Nikon microflex modelAFM automatic photomicrographic attachment.

Quantification of intravenously injected bacte-ria in mice. Mice injected with 1010 viable S.typhimurium were killed by cervical dislocation after30 min. The liver and spleen were removed andhomogenized with a Teflon and glass homogenizer in10 ml of isotonic sodium chloride, and quantitativepour plates were prepared. The stomach, intestinaltract, skin, and tail were discarded. The remainingcarcass was homogenized in 100 ml of saline in a

Waring blender for 2 min and pour plates were alsoprepared.

RESULTS

Fate of viable S. typhimurium injectedintravenously or perfused directly into liversof CF-1 mice. Thirty minutes after intravenousinjection of 1.2 x 109 viable Salmonella intomice, over 80% of the bacteria had been killed(Table 1). Of the bacteria not killed, 8.2% were

recovered from the liver and a roughly equalnumber from the carcass. The spleen harbored2.8% of the remaining viable bacteria. Since theliver is well established as a major reticuloendo-

TABLE 1. Survival of S. typhimurium 30 min afterintravenous injection into mice

Organ Recovery (%) Killing (%)

Liver 8.2 1.OaSpleen 2.8 1.4Carcass 7.5 1.1Total 18.5 1.5 81.5

a Average value standard deviation from at

least six separate experimental determinations.

thelial reservoir for the clearance and killing ofintravenously administered bacteria, we as-sume that most of the bacteria were cleared andkilled in the liver.

After a single pass of 1.1 x 1010 bacteriathrough the perfused mouse liver (which hadpreviously been washed free of blood), slightlymore than 30% of the organisms were recoveredin the effluent, i.e., washed out by continuedperfusion with 50 ml of sterile M-199 for 30 min(Table 2). The washed livers were homogenizedin a Teflon and glass homogenizer, and thenumber of viable bacteria was determined.Column 2 of Table 2 shows that all of thetrapped bacteria were recovered, suggestingthat mouse livers, depleted of humoral factors,could efficiently trap but not kill bacteria.Phase micrographs of perfused mouse livers

(Fig. 1) revealed the presence of bacteria inmany of the liver sinusoids, but apparently theywere not phagocytosed. Figure 1A shows asection of normal perfused liver in the absenceof bacterial infusion. Figure 1B is a section ofliver perfused with a single pass of 1.4 x 1010bacteria. Organisms are beginning to fill sinu-soids. Figures 1C and 1D are from mouse liversperfused with 3 x 1010 bacteria. At this concen-tration organisms appear in a "log-jamming"pattern in the sinusoids and, although notkilled, are also not washed out by continual per-fusion with sterile M-199. Figure 2 shows anelectron micrograph of a cross-section of thepacked bacteria. The organisms are trapped butdo not appear to be phagocytosed. It should benoted that following bacterial infusion therewas no significant decrease in the flow rate ofmedia through the perfused livers except whenvery high concentrations of organisms (7 x 1010)were infused.

Clearance of intravenously injected bacte-ria by intact rats and by the perfused ratliver. To be certain that any in vitro modelapproximated in vivo realities, the clearancerate of intravenously injected S. typhimurium

TABLE 2. Clearance and killing of viable S.typhimurium by the perfused mouse liver

Recovery (%)

Effluent Homogenate TotalEx- Actual recovery

pected

31.4 + 8.3a 68.6 73.6 + 12.3 105.3 + 10.9

aAverage value 4 standard deviation from nineseparate experimental determinations. 1010 bacteriawere perfused.

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414 MOON, VRABLE, AND BROKA

. ..e-Z I-'A' %' - 1 - "P Iflf, s ] v

FIG. 1. Phase micrographs of mouse liver perfused with S. typhimurium (x400). (A) Normal perfused mouseliver. (B) Mouse liver perfused with 1.4 x 101° S. typhimurium. (C, D) Mouse liver perfused with 3.0 x 1010S. typhimurium.

in rats was determined. Figure 3 shows that by 2min, more than 60% of the bacteria were re-moved from the blood, and this clearance pro-gressed rapidly with more than 95% of theorganisms removed by 30 min.When the same number of bacteria were

infused directly into the portal vein of thewashed, perfused, rat liver in situ, over 70% ofthe bacteria were trapped in a single pass(Table 3). A similar percentage of bacteria wereremoved when approximately 10", 108, or 1010CFU was injected. Column 3 of Table 3 shows

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ANTIBACTERIAL FUNCTIONS OF PERFUSED LIVERS 415

~~~: K~~~~~Z,..

4

|* . *b '~~~I

FIG. 2. Electron micrograph of a cross-section of a liver sinusoid from a mouse liver perfused with 3.0 x 1010S. typhimurium (x 13,600).

that, at all three dose levels, most of the balanceof the bacteria not recovered in the effluentcould be accounted for as viable cells recoveredin liver homogenates.Because the perfused liver could trap 1010

bacteria as efficiently as 101 on a single pass, wetried to determine how many bacteria could be

infused before the trapping capacity of the liverwas exceeded. Administration of five doses of1010 bacteria to a liver over a period of 3 h withwashings of 100 ml of sterile M-199 betweeneach pass resulted in slightly increased trappingability by the perfused organ (Table 4). Afterthe fifth injection of 1010 bacteria, no signs of

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416 MOON, VRABLE, AND BROKA

saturating the liver's trapping capacity was

noted and there was no inhibition of flow ratethrough the organ. Electron microscope eval-uation of the livers after this procedure revealedessentially healthy cells (micrographs not pre-

sented).Bactericidal activity of the perfused rat

liver in the presence of whole blood or bloodcomponents. Table 5 describes the effects ofmixing blood or blood components with eitherbacteria alone or bacteria plus the perfusionmedium. With no additives, 6.9% of the bacte-ria were killed after 30 min (line 1). Continuedwashing of the liver for 3 h with M-199 aloneincreased the killing to 17.5% (line 2). Washingfor this extended period of time did not increasethe number of organisms in the effluent. When0.1 ml of the bacterial suspension was mixedwith 0.9 ml of whole blood obtained by cardiacpuncture from the donor rat immediately beforecatheterization of the liver, bacterial trapping

00.s

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fz4

(K

02 5 10 I; 30

TlIME IN M INUT ES

FIG. 3. Percentage of viable bacteria in blood ofrats at various times after intravenous injection of 101°CFU of S. typhimurium strain SR-1I. Heart bloodsamples were quantitated for viable bacteria at thetimes noted. Calculations were made assuming a 20-ml blood volume for the rat.

TABLE 4. Trapping of multiple passes of S.typhimurium by perfused rat livers

Pass Recovery Trappedbno.' in effluent (%c) (%)

1 28.1 + 10.4c 71.92 16.9 + 4.1 83.13 13.5 ± 3.5 86.54 12.3 ± 3.9 87.75 10.3 ± 3.7 89.7

a Each pass contained approximately 1010 CFU.bDetermined by subtracting the percentage recov-

ered in the effluent from 100%.c Average value + standard deviation.

or killing by the perfused liver was not enhanced(line 3). When the whole blood was mixed withthe wash medium at a dilution of 10 ml of wholeblood to 90 ml of M-199 in addition to bloodbeing present in the bacterial suspension per se,trapping was enhanced about 10% but now over50% of the trapped bacteria were killed, asshown by a reduction in recovery of live bacteriafrom the liver homogenate (line 4). Preliminarystudies showed that mixing blood or plasmawith bacteria had no bactericidal effect (datanot shown). Plasma was as effective as wholeblood in this system (line 5), whereas thecellular elements of the blood did not altertrapping or increase bacterial killing (line 6).

DISCUSSIONThis study describes a unique experimental

model which can distinguish between the bacte-rial trapping and bactericidal properties of theliver. Table 2 shows that the perfused mouseliver has both a large affinity and capacity totrap viable S. typhimurium on a single pass.The micrographs in Fig. 1 make it evident thatthis trapping does not exclusively reflect phago-cytosis. Instead, the bacteria appear trapped insinusoids and if high enough concentrations areadministered, they are stacked in the vesselsgiving a "log-jam" appearance. Continued

TABLE 3. Trapping of a single pass of viable S. typhimurium by the isolated perfused rat liver

Recovery (%)No. of bacteria Effluent Homogenate Total Killing

perfused recovery (%

Expecteda Actual

2.3 x 106 38.4 ± 8.6 61.6 60.2 + 10.7 98.6 ± 5.8 1.42.3 x 108 27.8 ± 7.4 72.2 65.4 + 8.4 93.2 ± 7.4 6.89.6 x 109 21.1 ± 5.8 78.9 72.0 i 7.9 93.1 ± 7.2 6.9

a Determined by subtracting the percentage recovered in the effluent from 100%.Average value ± standard deviation of at least seven separate experimental trials.

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ANTIBACTERIAL FUNCTIONS OF PERFUSED LIVERS 417

TABLE 5. Effect of whole blood, plasma, and blood cells on trapping and killing of S. typhimurium by theperfused liver of normal rats

Recovery in Recovery from TotalKillingExperimental effluent homogenate recovered Kilin

No additives 21.1 5.8a 72.0 7.9 93.1 7.2 6.9No additives; extended perfusion 11.6 ± 4.2 70.9 ± 17.4 82.5 ± 20.0 17.5Whole blood added to only bacteria 13.9 ± 5.1 81.4 ± 8.5 95.3 ± 7.7 4.7Whole blood added to bacteria and wash 11.2 ± 3.4 31.0 ± 14.5 42.2 ± 15.8 57.8Plasma added to bacteria and wash 7.3 ± 8.9 41.3 ± 11.5 48.6 ± 18.5 51.4Blood cells added to bacteria and wash 17.0 i 0.5 82.0 ± 1.25 99.0 ± 1.5 1.0

a Average value ± standard deviation of at least six separate experimental trials.

washing of the liver for periods as long as 3 hwith sterile M-199 does not result in significantrelease of these organisms from mouse liver(data not shown). Before bacteria were infusedinto the liver, most gross blood had been washedout and thus Table 2 also shows that in theabsence of humoral factors only negligible kill-ing occurs after 30 min. This is in sharp contrastto the intact mouse where over 80% of intrave-nously injected organisms were killed in thesame time period (Table 1).The similarities in trapping (clearance) but

differences in killing between the in vivo and insitu mouse systems led us to suspect that bloodelements may play a more significant role inkilling than in trapping. Such a distinctioncannot be made with in vivo models whereblood and liver are always mixed, and hence theperfusion model represents a unique experimen-tal opportunity to explore this possibility. Themouse was not as optimal an experimentalanimal as the rat for investigating the relation-ship between these two parameters since largeamounts of homologous blood could not beobtained from the mouse. Hence, we shifted ourstudy to the rat.

It is evident from previous studies (18), aswell as our data, that the rat liver has a verylarge capacity for trapping bacteria. Even after5 x 101° S. typhimurium had been perfused, noevidence such as decreased perfusion flow ratesor extensive clogging of the sinusoids withbacteria was observed even though numerousbacteria were routinely visualized in the sinu-soids (rat micrographs not presented). By con-trast, in the mouse model after a third injectionof 1010 bacteria, the liver flow rate slowed andthe livers swelled, suggesting that the sinusoidsof this smaller organ were so clogged withbacteria that continued perfusion was impossi-ble. We have also demonstrated that the trap-ping phenomenon is not unique to S.typhimurium since Escherichia coli, Staphylo-

coccus aureus, and C. albicans are all avidlytrapped as well as or better than Salmonella(unpublished data).A comparison of the data in Fig. 2 and Table

3 shows that the bacterial clearance rate after asingle pass of organisms through the perfusedrat liver (approximately 70%) roughly approxi-mates the clearance rate of intravenously in-jected bacteria after 2 min. The major distinc-tion between these two systems is the absence ofgross blood in the in situ perfusion. Addition ofeither fresh whole blood or plasma to thebacteria and perfusion medium only slightlyenhanced trapping but increased killing over400%. It must be emphasized that the humoralelements had to be present in both the bacterialculture and the wash medium before significantkilling occurred. Mixing blood with bacteria butnot the wash media did not enhance killing (cf.Table 5). Since no significant killing occurredwhen the bacteria plus blood or plasma wereperfused through the apparatus in the absenceof liver, it is evident that all three componentsof the system (i.e., blood, liver, and bacteria)are needed for significant killing to occur. Datafrom our laboratory (Zlydaszyk and Moon,manuscript in preparation) and others (18)demonstrate that even a septicemic animal hasa very large capacity to clear bacteria from thebloodstream. Hence, it is difficult to under-stand why livers fail to clear more bacteria thanthey do during infectious septicemia. Conceiva-bly, as suggested by Saba (19), humoral opsonicfactors may be quantitatively more importantthan cellular reserves in limiting phagocytosis.If this is true, then current problems such as theenhanced susceptibility to infections in immu-nosuppressed patients (3, 12, 13, 17, 21) mayreflect not just decreases in cellular phagocyticcapacity but also depletion of humoral factorsnecessary for bactericidal activity. The perfusedliver system offers a unique experimental oppor-tunity to explore these relationships.

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418 MOON, VRABLE, AND BROKA

ACKNOWLEDGMENTS

This investigation was supported by Public Health Servicegrant AI-09394 from the National Institute of Allergy andInfectious Diseases.

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