Clin. exp. Immunol. (1969) 5, 173-187.

THE ACTIVITY OF MOUSE KUPFFER CELLSFOLLOWING INTRAVENOUS INJECTION OF

T4 BACTERIOPHAGE

C. J. INCHLEYDepartments ofZoology and Surgical Science,

Edinburgh University, Scotland

(Received 16 January 1969)

SUMMARY

The response of macrophages from the livers and spleens of mice given a singleimmunizing dose of T4 bacteriophage has been studied. Following their rapidremoval from the circulation, phage particles were found to be concentrated in theliver to a level twelve times that for the spleen. Investigation of the fate of ingestedphage showed that it was disposed of more rapidly in the liver than in the spleen,as measured by the disappearance of viable T4 particles and by the loss of radio-active label following injection of [131I]T4. It was also found that antigen-containing Kupffer cells could elicit little or no antibody synthesis on transfer intonormal syngeneic recipients, or on incubation with lymphoid cells in vitro. It issuggested that these macrophages differ from other components of the reticulo-endothelial system in their treatment of T4 antigen, and may be concerned mainlywith its breakdown and disposal rather than with providing a stimulus for theinitiation of antibody synthesis.

INTRODUCTION

The intravenous injection of particulate antigen is followed first by its rapid clearance bythe reticulo-endothelial system (RES), and then by the production of specific antibody.There is an increasing amount of evidence which suggests that these two events may beinter-related, and that RE cells possess the ability to participate in the initiation of theantibody response (Fishman, 1961; Adler, Fishman & Dray, 1966; Ford, Gowans &McCullagh, 1966; Gottlieb, Glisin & Doty, 1967; Unanue and Askonas, 1968b; Mitchison,1969). This conclusion is dependent in part, however, on information from in vitro systems,and while peritoneal exudate cells have been widely used in these studies, it has to bedetermined whether macrophages from other sources act in a similar way.A number of reports have suggested indirectly that other populations of macrophages

Correspondence: Dr C. J. Inchley, Department of Medicine, New York University Medical Center, 550First Avenue, New York, N.Y. 10016, U.S.A.

173

174 C. J. Inchley

possess such an ability (Garvey & Campbell, 1956; Ishizaka, Campbell & Ishazaka, 1960;Frei, Benacerraf & Thorbecke, 1965; Rolfe & Sinsheimer, 1965), and that intravenouslyinjected antigen which localizes in the RES may play some role in the induction or mainten-ance of the immune response. However, the study of Franzl (1962) indicated that if macro-phages are involved in the production of antibody, then this may be the prerogative of thosesited in lymphoid tissues only. Elsewhere their function may be quite different.The experiments described here were designed to investigate the role of Kupffer cells in

the treatment of bacteriophage antigen during the inductive phase of the immune response.The localization, loss of viability and catabolism of coliphage T4 within these cells has beenstudied in normal and X-irradiated mice and compared to that in the spleen. In addition,antigen-laden Kupffer cells from immunized mice have been transferred to syngeneicrecipients in order to determine whether they carried any immunogenic stimulus whichwas available to lymphocytes and which could, therefore, be detected by the appearance ofantibody in the hosts (Gallily & Feldman, 1967; Mitchison, 1969).

MATERIALS AND METHODS

AnimalsAdult CBA strain inbred mice were used throughout. For each experiment, mice of the

same age and sex were chosen, with a weight range of 18-25 g.

AntigenBacteriophage T4, grown and titred on Escherichia coli B, was used as antigen. Phage

stocks were maintained in 0-1 M-ammonium acetate at 40C and dilutions were made asrequired in 0.900 saline with the addition of thymol and gelatin (Adams, 1959).

ImmunizationMice were injected intravenously (i.v.) with a single dose of 5 x 108 plaque-forming units

(PFU) T4 in 0 I ml saline. This was found to give a near optimum antibody response withKmax of 15-25 (see Inchley & Howard, 1969).

IrradiationMice were restrained in polystyrene boxes 75 cm from the source and irradiated at a rate

of 66-4 r/min with a Westinghouse unit run at 230 kV and 15 mA. Filtration was 0 5 mmcopper and 1b0 mm aluminium.

Estimation of antibodyMice were bled from the retro-orbital plexus with a capillary tube, and their sera stored

at -20'C. Antibody assays were based on the technique of Adams (1959). A volume of0 05 ml of appropriate dilutions of serum from individual mice and a standard T4 sus-

pension were incubated together for 60 min at 370C. Following dilution in 10 ml saline toarrest the reaction, 0 1-ml aliquots were plated out in duplicate by the double layer method.Two control suspensions containing phage only were prepared and each plated out intriplicate without incubation (T4 was found to be stable in normal serum under these assayconditions).

Activity of Kupffer cells 175

Phage neutralizing activity is expressed in terms of the serum inactivation constant, K,according to the formula (Adams, 1959):

D P0K= 23-xlog-°

t P

where D is the final serum dilution, t the time of incubation in minutes, and P0 and P thenumber of PFU before and after incubation. K values of 0'006 and over (= 15% neutraliza-tion at a 1:2 serum dilution) are held to represent significant activity at the 10% level.

Isotopic labelling of T4Labelling with " Cr was carried out as follows: 250 pc isotope in sodium chromate, pH 8 0

(Radiochemical Centre, Amersham), was added to 0 1 ml of a T4 suspension containing1 2 x 1012 PFU/ml. The volume was made up to 2-0 ml with phosphate buffered saline atpH 8-0 and incubated for 30 min at 370C. The mixture was then dialysed againstbuffer for 48 hr, after which time 1-10% of the activity was associated with the phage. Lessthan 1% of this amount represented unbound 51Cr.

Labelling with 131I was based on the method of McFarlane (1958). A volume 0-02 mliodine monochloride containing 0-42 mg I/ml, and 0-025 ofa suspension ofT4 (1012 PFU/ml)were each adjusted to pH 8*0 in an equal volume of phosphate buffer and added in turn to200 pic carrier free "3'I in less than 0*01 ml N/50-NaOH (Radiochemical Centre, Amersham).Following 3 hr incubation at room temperature, the mixture was dialysed against 500 mlsaline at 4°C for 24 hr. An average of 1.5% of the initial activity was associated with therecovered phage, of which 4.700 remained unbound. Mice were given iodide water (100 mgNal/litre tap water) ad libitum for 24 hr before injection of [131I]T4.

Preparation of Kupffer cell suspensionsLiver cells were isolated by a modification of the collagenase and trypsin digestion method

of Garvey (1961) as described by Howard et al. (1965). The yield from normal mouse liverswas 1 0-1.5 x 107 cells. The results of differential counts on three preparations are given

TABLE 1. The composition of liver cell suspen-sions isolated from CBA mice

% cell type in eachsuspension

Cell types scored1 2 3

Macrophages 74.4 71P6 81-0Large lymphocytes 10-8 9-2 9.3Small lymphocytes 5-0 5 6 1-3Eosinophils 1-0 07Neutrophils 3-8 6-0 2-0Basophils 0 3Parenchymal cells 4-4 6-2 4-3Others 1-6 04 1.1Total cells scored 500 500 300

176 C. J. Inchley

in Table 1, from which it will be seen that 70-85% of the cells were Kupffer cells, and thatthe contamination with small lymphocytes and parenchymal cells was relatively small.Viability was greater than 950 as measured by trypan blue exclusion.

Preparation of lymph node cell suspensionsThe cervical, axillary, brachial and inguinal nodes were removed from normal mice,

gently broken up together in medium 199 in a loose-fitting ground glass homogenizer, andthe resulting suspension filtered through a stainless steel sieve. The cells were washed threetimes in medium 199 and made up in this medium to a suitable concentration. Cell viabilitywas greater than 9400.

RESULTS

Uptake of T4 bacteriophage by the RESThe work of Brunner et al. (1960) has indicated that virus particles are cleared from the

mouse circulation at rates similar to those found for other particulate materials (Benacerrafet al., 1957, 1959a; Biozzi et al., 1960). Rapid uptake ofT4 by the mouse RES was establishedby plating out dilutions of blood withdrawn at intervals from groups of three to four micefollowing i.v. injection of 5 x 108 PFU. The results for two of these groups are presented inFig. 1.

8-0

0Z0 6-0-

U_4-0-

2-0

0 2 3 4 5 6 0 2 3 4 5 6Hours after injection

FIG. 1. Clearance of T4 in normal mice: Mean concentrations of plaque-forming units (PFU)in the circulation of two groups of mice at intervals following injection of 5 x 108 PFU T4 i.v.Vertical lines represent the range at each point.

The great majority of phage (more than 990%) was phagocytosed during the first 30 min.Clearance continued at this rate up to 1 hr after which a prolonged phase of slower elimina-tion commenced. By 6 hr approximately 104 PFU/ml blood could still be recovered; at48 hr this figure was reduced to 2-7 x 102. In one instance particles were isolated from theblood as late as day 5. If the rate ofclearance during the first 30 min is taken to be exponential,then it can be expressed for each group by the phagocytic index K (Biozzi, Benacerraf &Halpern, 1953; Biozzi et al., 1960). Calculation of these values gives Ka = 0-076 andKb = 0-082, indicating a slower rate than that observed in mice for 32P-labelled vesicularstomatitis virus (Brunner et al., 1960). E. coli (5 x 108/dose) and S. enteritidis (107-8 x 109/

Activity of Kupffer cells

20 g) are also cleared more rapidly than 5 x 10' PFU T4 (Benacerraf et al., 1959a; Biozziet al., 1960).The location of phagocytosed material was determined 18 hr after i.v. injection of 5tCr-

labelled phage. The results in Table 2 confirm the efficiency of the liver in removing T4 fromthe circulation-70-9000 of the recovered activity was located in this organ. The mean valuefor the liver was nearly twelve times that for the spleen, which contained less than 10% ofthe counts in all but one animal. Small amounts of label were also detected in other tissues.

TABLE 2. The distribution of [5'Cr]T4 in the tissues of the mouse

InguinalMouse Liver Spleen Kidneys Lungs lymph %Y dose

nodes recovered

1 77-8 5 4 6.1 6-8 3.9 89-62 81*4 6-0 6-3 3-3 3-0 69-63 81-2 7-6 4-7 3-1 3.4 87-94 86-8 5.2 3-1 1-7 3-2 8095 71-7 12-2 9-7 2-1 4 5 73-26 85 2 6-1 3.3 3-5 1-9 99.3

Mean % recovered 80-7 7-1 5.5 3-4 3-3 83-4counts

Activity in each organ is expressed as a percentage of the total recovered counts.

Inactivation of T4 in the livers and spleens of normal miceThe spleens and perfused livers of mice injected with T4 up to 21 days previously were

broken up in a close fitting Teflon homogenizer so as to disrupt their constituent cellswithout damaging ingested bacteriophage. Dilutions of these homogenates were platedand the number of viable phage particles in the whole organs calculated. Fig. 2 records theresults up to day 14, the latest time at which PFU could be recovered from the liver. Whenthe number of remaining PFU was small a limitation was imposed by the volume of un-diluted homogenate which could be plated. Therefore, when no particles were detected anestimate was made of the number which might have escaped detection, and these points areindicated by hollow symbols.

Following initial phagocytosis, there was an immediate and rapid decrease in the numberof PFU which could be recovered from both liver and spleen. Although T4 was still detect-able in these organs on days 5-7 (and in one instance in the liver on day 14), inactivation ofingested phage continued until the plating out of relatively large volumes of undilutedhomogenate failed to reveal plaques.

It was found, however, that the number of PFU which could be recovered from thespleen 24 hr after injection was greater than that found in the liver. This difference persistedat least until day 5, at which time there were as many as fifty times more PFU in the spleen.Since the number of particles which originally localized in the liver was twelve times that forthe spleen, these results imply that inactivation of antigen occurs much more rapidly withinKupffer cells than within splenic macrophages.

177

178 C. J. Inchley

20~

60

4-0 - !

2-0 =-

0 2 4 6 8 10 12 14Days after injection

FIG. 2. Inactivation of T4 in the livers (a) and spleens (b) of normal mice following injection of5 x 108 PFU i.v. Each point represents the mean of three animals. For explanation of opensymbols, see text. ----, Experiment 1 (liver only); , Experiment 2; ---,Experiment 3.

The effect of irradiation on phagocytosis and inactivation of T4In order to determine whether the events described above were radiosensitive, in connec-

tion with studies on the fate of T4 as immunogen in irradiated mice (Inchley & Howard,1969), similar experiments were carried out in mice exposed to doses of 450 and 600 r 24 hrbefore administration of phage. Fig. 3 shows the clearance of T4 in groups of mice sotreated. It was found that neither dose of X-rays impaired the ability of the RES to removephage particles from the circulation. The initial phagocytic indices (K = 0-053 after 450 rand 0-082 after 600 r) were similar to those found for normal mice. At 1 hr the number of

8.0 (a) (b)

060

0~

40

- 2-0

0 2 3 4 5 6 0 2 3 4 5 6Hours after injection

FIG. 3. Clearance of T4 in irradiated mice: Mean concentration of PFU in the circulation ofmice given 450 r (a) or 600 r (b) 24 hr prior to injection of T4. Vertical lines represent the rangeat each point. For control curve, see Fig. 1 (Group A).

Activity of Kupffer cells 179

circulating PFU in both groups had decreased to around 105/ml blood, and thereafter therate of clearance became slower. By 6 hr the mean concentrations in both normal andirradiated animals were about 104 PFU/ml blood.Mice exposed to 450 and 600 r were also killed in groups of three at intervals up to 7 days

after the injection of T4, and their spleens and perfused livers assayed for surviving phage(Fig. 4). The numbers of PFU which could be recovered from these organs were similar tothose observed for normal animals, with the exception that the mean values for the spleenat 1 day were ten times less in irradiated mice. PFU could still be detected in all livers andspleens at the end of the 7-day period. Although at day 1 there were twice and ten times asmany PFU in the livers of mice given 450 and 600 r, respectively, subsequently at least tentimes more PFU were found in the spleen than in the liver in both groups. Irradiation withthese doses does therefore not obscure the faster rate of phage inactivation within Kupffercells.

(a) (b)

6-0-

0

- 0

0 1 2 3 4 5 6 7 0 2 3 4 5 6 7Days after injection

FIG. 4. Inactivation of T4 in the livers (a) and spleens (b) of irradiated mice. Each pointrepresents the mean of three animals. For control curves, see Fig. 2 (Experiment 2).450 r; ----, 600r.

Degradation of 13'I-labelled T4 in the livers and spleens of normal miceSupport for the contention that liver and spleen macrophages degrade antigen at different

rates was also obtained from mice injected with "1'I-labelled phage. The elimination of the"1'1-label from the tissues is held to be an accurate indication of the breakdown of theprotein to which it is attached. In these experiments, the amount of radioactivity remainingin the liver and spleen was measured at intervals up to 7 days after administration oflabelled T4. At times up to 6 hr, fifteen mice were killed for each determination, while atlater times six or nine mice were used. The results are given in Fig. 5, where the amountof retained label is expressed as a percentage of the injected phage-bound activity.The "1'I-label was very rapidly lost from Kupffer cells, and at 30 min, when less than 1%

of the dose remained in the circulation, only 310% remained in the liver. This rate of degrada-tion continued for up to 3 hr and started to decrease. There was relatively little further lossof label after 24 hr. Loss of activity from the spleen appeared to follow a different pattern.No overall decrease in activity was seen to occur between 15 min and 6 hr after injection,when the mean per cent levels were 2-5 and 2-8, respectively, and although there was some

180 C. J. Inchley

(a)30_

20

0

-o

40--o0o

0._1

20 \

0 2 3 4 5 6 7Days after injection

FIG. 5. Degradation of [13 I]T4 in the livers (a) and spleens (b) of normal mice. Each point isthe mean of six to fifteen determinations.

fluctuation of spleen counts during this period, the general picture agrees with previousobservations on the degradiation of 13"'-labelled S. enteritidis in mice (Biozzi et al., 1960).It seems clear from both studies that the initial degradation of ingested antigen may pro-ceed at different rates in the liver and spleen.

The effect of transferring Kupifer cellsfrom immunized to normal miceAs liver and splenic macrophages appeared to differ with respect to their treatment of T4

phage, it was desirable to test the ability of antigen-charged Kupffer cells to stimulate anti-body formation on transfer to normal syngeneic recipients. It seemed likely that any suchactivity would be apparent soon after exposure to antigen, and before the first appearance ofantibody. Accordingly, groups of mice were killed at 6, 12 and 24 hr after injecion of T4,and suspensions of their Kupffer cells prepared. Recipient mice were injected intraperiton-eally with doses of 0 9-2 0 x 107 cells, while control animals received an equal number ofcells which had been killed by heating at 490C for 20 min or disrupted by homogenization.All recipients were bled once, 10 days after cell transfer, and their sera assayed for anti-T4activity.The amount of antibody formed in ten groups of recipients is given in Table 3. Very little

immunogenic activity could be demonstrated either on the part of whole cells or of materialwhich was released when the cells were killed, and was only found at significant levelsfollowing 6- and 12-hr transfers. There was no evidence to support the contention thatdigestion of antigen by Kupffer cells produces hyper-immunogenic material (Garvey &Campbell, 1957). The results for live and killed cells were very similar and indicate that theability of Kupffer cells to initiate any sort of response coincided with the presence withinthem of relatively large amounts of undigested phage.

Kupffer cell suspensions were routinely checked for the presence of extracellular PFUwhich might have been released from cells damaged during isolation. In no instance was

Activity of Kupffer cells 181

TABLE 3. Anti-T4 activity in the sera of recipient mice 10 days after injection of live(group A), heat killed (group B) or homogenized (group C) liver cells isolated

6-24 hr after donor immunization

Time of Mean K valuestransfer Experiment Liver cell

(hr) dose (x 10') Group A Group B Group C

6 1 1-0 0010 (6) 0016 (6)2 1.0 0-027 (6) 0030 (6)3 2 0 0 015 (6) 0-022 (4) 0-018 (5)

12 4 0 9 0 010 (6) 0-016 (6)5 1-0 0 (6) 0007 (6) -6 1-0 0-011 (6) 0-014 (6)7 1.0 0 (9) 0 (9)8 1-5 0-015 (6) 0-020 (6) 0-013 (6)

24 9 1 0 0001 (4) 0001 (5) 0002 (5)10 1-0 0 005 (9) 0 005 (9) -

Control K 19-34 (11)(Day 10 K in normal mice immunized with 5 x 108 PFU T4)

The number of animals in each group is shown in parentheses.

there a sufficient number to immunize normal mice (less than 5 x 105/dose). It may be,however, that in combination with partly degraded phage fragments they were able toinduce the low levels of antibody found.

Kupffer cells were also prepared from mice immunized 4 and 7 days previously, by whichtime there was a high level of circulating antibody. In addition to recipients of killed cells,control groups also included X-irradiated (450 r) recipients of live cells to confirm that noantibody synthesizing capacity was transferred. No free T4 PFU were detected in thesesuspensions. Mice were bled as before, 10 days after transfer. The results are given in Table 4.

TABLE 4. Anti-T4 activity in the sera of recipient mice 10 days after injection withlive (group A) or killed (group B) liver cells isolated 4 and 7 days after donor im-

munization

Time of Mean K valuestransfer Experiment Liver cell(days) dose (x 107) Group A Group B Group C

4 11 1-0 0 008 (8) 0.002 (4)*7 12 0 9 0 (6) 0 (6)

13 09 0-004 (3) - 0006 (3)14 1-4 0 (3) 0 (3)15 1.0 0 005 (7) 0 005 (7)t

Live cells were also transferred into irradiated mice (group C). The number ofanimals in each group is shown in parentheses.

* Killed by heating at 490C.t Killed by homogenization.

182 C. J. Inchley

Again, anti-T4 activity was small or absent. The only significant neutralization of phagewas found in recipients of cells isolated 4 days after donor immunization. As tested bythis technique, therefore, the development of the immune response to T4 is not accompaniedby the accumulation within Kupffer cells of hyper-immunogenic products of antigendegradation.

An attempt to stimulate Kupffer cells with T4 in vitroIt could be argued that should macrophages bear an immunogenic 'message' at or upon

their surface (White, 1963; Nossal, Ada & Austin, 1964), then in vitro digestion of liverfragments by proteolytic enzymes might damage or destroy this property. The cells wouldthen be rendered immunologically inert. An experiment was therefore carried out in whichKupffer cells were first exposed to antigen in vitro, after their isolation from the liver.Freshly prepared suspensions in medium 199 were divided into 5-ml aliquots each con-

taining 6-0 x 106 cells. After preliminary incubation at 370C for 30 min, 6 x 108 PFU T4freshly suspended in 1 ml medium 199 were added to each culture and incubation continuedfor a further 90 min. At this time the supernatant was removed and the remaining cellsgently washed four times with 5 ml medium 199. Examination of the culture dishes revealedadhered cells which were later shown to be macrophages by the ingestion of neutral reddye (Howard, Boak & Christie, 1966). They were found to have removed more than 95%Oof the T4. To each culture was then added 4 x 108 lymph node cells in 2 ml medium 199.

TABLE 5. Anti-T4 activity in recipients of 5 x 107lymph node cells following their incubationwith antigen-rich Kupffer cells (groups A, B

and C) or with antigen alone (group D)

Mean K values inNo. of sera at:

Group miceDay 10 Day 38

A 5 0005 0B 6 0-002 0C 3 0 0D 4 0008 0

The cells given to the group C mice werekilled by heating before injection.

A culture containing lymph node cells and T4 only was also set up. All cultures were in-cubated for 3 hr, when the unattatched cells were harvested, washed three times and re-suspended in medium 199. Recipient mice were injected i.v. with 5 x 107 live or heat-killedcells, with less than 500 free PFU. They were bled 10 days later and again after a further28 days. The activity of their sera is given in Table 5.

It is clear that Kupffer cells exposed to T4 in vitro and then incubated with lymph nodecells were unable to stimulate antibody synthesis in the lymphocyte populations, as judgedby the titres in recipient mice (groups A and B). It was also found that lymph node cells

Activity ofKupifer cells

exposed to T4 alone (group C) synthesized little or no antibody. Since the intravenousinjection of antibody-producing cells into syngeneic recipients does not interfere with theirability to continue this activity (Cochrane & Dixon, 1962) it may be assumed that thein vitro conditions were not suitable for the induction of immunologically competent cells.The results are in accord with the observations that antigen-charged liver macrophageswere unable to initiate the production of antibody against T4 in vivo, and may reflect theinability of these cells to participate in immune responses to this class of antigen.

DISCUSSION

Clearance of a wide range of foreign materials from the circulation is effected mainly byliver macrophages and, to a lesser extent, by those of the spleen. Previous studies on the fateof virus particles in mice have shown that they conform to this pattern of localization(Erickson, Armen & Libby, 1953; Brunner et al., 1960), and the present work has establisheda similar distribution for bacteriophage T4.The possibility that Kupffer cells might also be capable of playing an essential role in the

immune response was first suggested by Garvey & Campbell (1957). Following injection of[33S]BSA into rabbits, these authors showed that the label persisted in the liver to 140 days,and that its loss at that time was at a rate which suggested it remained for several years.Further, it was found to be associated with RNA, and to have maintained or improved itsimmunogenicity. These observations gave rise to the hypothesis that antigen fragmentsmight control the whole course of the immune response (Campbell, 1957). More recently,studies on peritoneal exudate (PE) cells have confirmed that the immunogenicity of variousantigens may be greatly increased following phagocytosis (Askonas & Rhodes, 1964;Gottlieb et al., 1967; Unanue & Askonas, 1968a; Mitchison, 1969), and may result in aproduct capable of stimulating lymphocytes to make antibody (Fishman, 1961; Friedman,Stavitsky & Solomon, 1965; Adler et al., 1966; Gottlieb et al., 1967). Other work involvingwhole cells rather than cell fractions has established the ability of antigen-charged peritonealcells to transmit antigenic information to lymphoid cells in vitro (Ford et al., 1966) andin vivo (Gallily & Feldman, 1967; Argyris & Askonas, 1968; Unanue & Askonas, 1968b;Mitchison, 1969). What is not certain, however, is whether the events described for PEcells are common to all macrophages, particularly as they are heterogeneous with respectto a number of other properties (Benacerrafet al., 1959a; Franzl, 1962; Mouton et al., 1963;Pavillard, 1963; Leake, Gonzalez-Ojeda & Myrvik, 1964). Following i.v. injection of antigenit might be expected that macrophages in the spleen would behave in a similar manner, dueto their close association with lymphoid cells (Nossal et al., 1966). On the other hand, theobservations reported here indicate the Kupffer cells may treat antigen in a different way.

[5"Cr]T4 was found to localize in the liver to a level nearly twelve times that for thespleen. In contrast with this initial distribution, it was subsequently found that more PFUcould be recovered from the spleens of normal mice up to 5 days after injection of phage.The slower rate of phage inactivation in the spleen proceeded from shortly after it had beenphagocytosed. It seems that liver macrophages are better equipped to inactivate particulateantigen, and that their main function may be to dispose of ingested material as quickly aspossible. Similar results were obtained in irradiated mice, although the difference in phageinactivation between the two organs was not so pronounced until after the first 24 hr.Sub-lethal X-irradiation appears not to impair macrophage morphology or phagocytic

F

183

184 C. J. Inchley

activity, although catabolism of antigen by PE cells may be altered (Brecher et al., 1948;Donaldson et al., 1956; Muramatsu, Morita & Sohmura, 1956; Gallily & Feldman, 1967).However, it has been shown that less antigen localizes in the spleen of irradiation animals,a deficiency which is compensated by increased Kupffer cell activity (Benacerraf et al.,

1959b), and this observation may explain the early difference between normal and irradiatedmice which was observed for T4.The contention that Kupffer cells and splenic macrophages degrade antigen at different

rates is further supported by the findings in mice during the first 6 hr after injection with[131I]T4. Whereas there was an immediate and rapid loss of label from the liver, phagesaccumulated and were broken down more slowly in the spleen. By 6 hr, only 16-9% of thedose radioactivity remained in the liver, a level six times that in the spleen at the same time.During this early period, therefore, Kupffer cells inactivated phages four times as fast as

splenic macrophages, and degraded T4 protein twice as rapidly. At later times PFU con-

tinued to be lost from both liver and spleen, while loss of label from both organs becameprogressively slower. It seems likely that phage particles were becoming fixed within some

sub-cellular unit rather than being completely broken down.During a study on the fate of "1'I-labelled S. enteritidis in mice, a similar pattern of

degradation to that observed for ['1I]T4 was found in the liver and spleen (Biozzi et al.,

1960). Similar results have also been reported for the breakdown of "1'I-labelled heat-aggregated human serum albumin (HSA) in rats (Gabrieli et al., 1965). The key study ofFranzl (1962) has indicated that differences in the manner in which antigen is processedor degraded may be reflected in its immunological function. He fractionated livers andspleens from mice immunized wth sheep erythrocytes and was able to demonstrate an

immunogenicity within spleen fractions, particularly lysozomes, which was entirely absentfrom liver preparations.

In the present study, an attempt was made to determine whether Kupffer cells developedany immunogenic activity following phagocytosis of T4. As judged by their relative inabilityto stimulate antibody in recipient mice, antigen-charged Kupffer cells are unable to particip-ate in the immune response from a few hours until 7 days after T4 injection. Although insome instances minute amounts of antibody were detected, especially when the concentra-tion of undegraded phage in the transferred cells was high, at no time did the responsecorrespond to the quantity of phage which localized in the liver. Even if it is assumed thatup to 9000 of Kupffer cells were lost during isolation, there would still remain sufficientantigen (ca. 3.75 x107 PFU) to give an antibody response with Kmax = 2-05-0.

It could be argued that Kupffer cells were not tested soon enough after exposure to antigen.In Fishman's experiments, PE cells were left in contact with antigen for only 30 min (Adleret al., 1966). In addition, Argyris & Askonas (1968) found that PE cells collected later thantwo hours after exposure to antigen became less effective with time in inducing antibody innormal recipients. That Kupffer cells can not be transferred sooner is in part a fault of thelengthy isolation procedure. However, no antibody was made by lymphocytes incubatedwith Kupffer cells after their exposure to T4 for 90 min in vitro. The negative findings can notsimply be attributed to the dose size or intraperitoneal route of injection of Kupffer cells,as essentially similar studies with PE cells have yielded positive results (Russell & Roser,1966, Gallily & Feldman, 1967; Argyris & Askonas, 1968). Nor can they be blamed on theenzymatic digestion of liver fragments. The isolated cells were not only viable, but able toadhere to glass surfaces and to ingest both antigen and neutral red dye. It has also been

Activity ofKupffer cells 185

found that Kupffer cells separated by this method are capable of fixing cytophilic antibody(Staines, personal communication). Although a hypothetical surface site for the fixation ortransfer of antigenic information might have been damaged during isolation, it is apparentthat a number of cell membrane properties remained intact.

It, therefore, appears that Kupffer cells are unable to take part in the induction of anti-body to T4, a conclusion which is supported by the finding that T4 localized in the livers ofsplenectomized mice was not able to stimulate replacement lymphoid cells to make antibody(Inchley & Howard, 1969). Kupffer cells may then differ from other macrophages withrespect to the treatment of particulate antigen, and the catabolic differences which werefound between the liver and spleen may reflect, if not be related to the immunologicalactivity of these organs. This distinction supports the earlierwork of Franzl (1962) with sheeperythrocytes. It may not, however, apply to soluble antigens as it has been found that theresponse to BSA in mice may be mediated as effectively by enzyme isolated Kupffer cellsas by peritoneal macrophages (Mitchison & Howard, personal communication). Theproblem remains as to the relationship of these events to immunogenic material which hasbeen observed in the liver at long intervals after the administration of antigen. This may beimportant for the maintenance of immunity. Alternatively, it may simply represent a by-product of the efficient disposal of foreign antigen by Kupffer cells.

ACKNOWLEDGMENTS

The technical assistance of Mr N. Anderson is gratefully acknowledged. I should also liketo thank Dr J. G. Howard for helpful discussion, and Dr N. A. Mitchison for facilitiesprovided at National Institute for Medical Research, London, N.W.7. Part of this work wassupported by Medical Research Council Grant 965/300/B.

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