Clin Exp Immunol 1994; 97:242-247

IgG-mediated phagocytosis in regenerated splenic tissue

M. T. R. CLAYER*, P. A. DREW*t, A. S.-Y. LEONGJ & G. G. JAMIESON* *Department of Surgery, TheUniversity of Adelaide, tSchool of Nursing, The Flinders University of South Australia, and t The Division of Tissue Pathology,

The Institute of Medical and Veterinary Science, Adelaide, South Australia

(Acceptedfor publication 26 April 1994)

SUMMARY

The risk of severe infections after splenectomy is well established. Operations such as auto-

transplantation, splenic artery ligation or partial resection have been advocated for the retentionor regeneration of splenic tissue following splenic trauma. The potential of such tissue to protectfrom infection is unclear. The ability of splenic tissue to phagocytose IgG opsonized syngeneicerythrocytes was measured in rats 6 months following splenectomy and splenic auto-transplantation, splenic artery ligation, total or partial splenectomy, and compared witheusplenic controls. In eusplenic and partially splenectomized rats 71% of the label was clearedat 3 h, compared with approximately 50% in rats following total splenectomy, splenectomy andsplenic autotransplantation or splenic artery ligation. The autotransplanted and the ligated splenictissue cleared less than 10% compared with control spleen, but there was no difference betweenthem when clearance was expressed as uptake per gram of tissue. Splenic autotransplants andligated spleens were small and histologically abnormal, with an increase in the red pulp,significantly less white pulp and marginal zone, and the frequent absence of a central arteriolein the white pulp. The clearance of label was proportional to the amount of red pulp in the tissue,although the red pulp from the regenerated tissues was not as efficient at phagocytosis as controlred pulp. The tissue which regenerated following autotransplantation or splenic artery ligation didnot result in greater clearance of erythrocytes from the circulation than that which occurred insplenectomized rats.

Keywords splenic autotransplantation Fc receptor phagocytosis IgG

INTRODUCTION

The spleen is an integral component of the reticuloendothelialsystem, with major roles in the mounting of antibodyresponses, and the phagocytosis and clearance of matter fromthe blood. A number of studies have clarified the specific natureof splenic phagocytosis [1, 2]. While IgM and C3b opsonizedparticles are removed from the circulation predominantly bythe liver [3], the spleen is required for the removal of IgG-coated particles from the blood, particularly those which arepoorly opsonized [4]. The spleen therefore is important indefence against bacteraemia, particularly when the patienthas low concentrations of specific antibody in the serum. Inthe absence of the spleen, clearance of bacteria opsonized withlow amounts of IgG will be impaired, and if the organismmultiplies rapidly, bacteraemia may quickly progress to thesyndrome of overwhelming post-splenectomy infection (OPSI).

Correspondence: Dr P. A. Drew, Department of Surgery, RoyalAdelaide Hospital, North Terrace, Adelaide, South Australia 5000,Australia.

This syndrome is characterized by sudden onset, rapid andfulminant course, frequent absence of a septic focus, unusuallyhigh bacteraemia, resistance to normally adequate therapy, anda high mortality rate [5].

The preservation of as much viable splenic tissue as possiblefollowing splenic trauma is now advocated in an attempt toprotect patients from the risk of OPSI. Splenorrhaphy, partialsplenectomy, splenic arterial ligation or autotransplantationhave all been advocated as techniques to retain functionalsplenic tissue and potentially avoid the problems associatedwith asplenia [6, 7]. Nevertheless, it is uncertain if theseprocedures adequately retain or restore the splenic functionnecessary to protect against OPSI. A number of patients havedied from OPSI despite the presence of large amounts ofsplenosis tissue [8, 9].

The purpose of this study was to compare the phagocyticcapacity of regenerated splenic tissue with that ofnormal tissue.The IgG-mediated phagocytosis of particles was measured incontrol rats, and rats which had undergone either totalsplenectomy, partial splenectomy, splenic artery ligation orsplenectomy with splenic autotransplantation.

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IgG-mediated phagocytosis in regenerated splenic tissue

MATERIALS AND METHODS

AnimalsPorton strain male rats approximately 250 g in weight from theCentral Animal House, University of Adelaide, Australia, wereused. They were fed a standard diet of rat pellets and water. Afemale New Zealand white rabbit was used for the preparationof antibody against Porton rat erythrocytes. The studies wereperformed in accordance with the guidelines and with theapproval of the Animal Ethics Committee of the Universityof Adelaide.

Surgical proceduresThe rats were allocated into one of five experimental groups,with 15 rats in each. The rats in the first group were splenecto-mized; in the second, hemisplenectomized; in the third, splen-ectomized and autotransplanted; in the fourth, the splenicartery was ligated; the fifth group served as unoperatedcontrols. All operative procedures were performed usinghalothane-nitrous oxide anaesthesia. For splenectomy, thespleen was mobilized via a midline incision, its vasculatureligated and divided, and then removed. Hemisplenectomy was

performed in the same manner, except that the vessels supply-ing only one half of the spleen were ligated and divided. Thedevascularized portion was excised, and the remainder with itsblood supply intact replaced into the abdomen. Splenic arterialligation was performed also in the same manner, except that thevessels were not divided after ligation. For autotransplantation,the spleen was removed and a piece of the splenic tissueweighing approximately 225 mg was sutured onto the mesen-

tery of the terminal ileum. All rats were allowed 6 months torecover from their operation.

Preparation ofIgG antibody to rat erythrocytesAntibody was prepared by injecting a rabbit subcutaneouslywith washed Porton rat erythrocytes emulsified in Freund'sincomplete adjuvant (FIA). The immunization was repeatedtwice, at monthly intervals. The IgG antibody fraction was

prepared by octanoic acid fractionation of the serum [10]. Theconcentration of antibody was determined by haemagglutina-tion (HA) assay, and the lowest dilution of the sample whichresulted in visible agglutination was assigned the value of a

100% HA dose.

Clearance ofIgG-opsonized erythrocytesSyngeneic rat erythrocytes were labelled with 51Cr as sodiumchromate. Packed, labelled erythrocytes and the dilution of theIgG antibody (or saline for the preparation of unopsonizedlabelled erythrocytes) were then incubated at 37°C withcontinuous slow mixing for 30 min, washed twice in salineand re-suspended as a 50% packed cell solution. The rat inwhich clearance was to be measured was anaesthetized, and theabdomen opened with a midline incision. A 100-jil aliquot ofthe labelled erythrocytes was injected into the inferior vena

cava. The syringe was counted before and after injection andthe amount of radioactivity injected determined by subtraction.In some of the rats blood samples were taken from the inferiorvena cava at intermediate time points before they were killed.All rats were killed 3 h after injection, and 2 ml of blood, thespleen, liver and lungs were harvested, weighed and the radio-activity in each counted. The counts in the blood measured the

amount of radioactivity not removed from the circulation. Theresults are expressed as the percentage of the total countsinjected, or the percentage of the total counts injected pergram of tissue (% ct/min per gram).

AutoradiographyIn five control rats the zone within the spleen in whichphagocytosis of the erythrocytes occurred was determined byautoradiography. Opsonized, labelled erythrocytes wereinjected intravenously, and 3 h later the spleen was excisedand fixed in formalin. Sections of 5 pm thickness were cut andplaced on photographic film (Kodak XOMAT AR5 GBX-2)for up to 4 weeks. The film was developed, and the spleensections were then stained with haematoxylin and eosin.

Histological examinationTissue was fixed in buffered formalin, and 5 Htm sections cut andstained with haematoxylin and eosin. The percentage of thearea occupied by white pulp (periarteriolar sheath and germinalcentres) and marginal zone was calculated for each specimenusing computer image analysis. The area of the red pulp was thetotal area of the section less that of the white pulp plus marginalzone. The relative amount of each compartment of the spleenwas estimated by multiplying its percentage area by the weightof the tissue [11]. All areas of white pulp in a section (to amaximum of 50) were examined, and the number with a centralarteriole was expressed as a percentage of the total.

Statistical analysisAll results are reported as the median and range of the group.Groups were compared using the Mann-Whitney U-test.Correlations between groups were measured using the Spear-man rank sum correlation test.

RESULTS

Effect of the amount of antibody on splenic clearanceThe dilution of the IgG used for opsonization which mediatedrelatively greater splenic clearance compared with the liver wasdetermined in controls rats. Labelled erythrocytes wereopsonized with one of four dilutions of IgG: 100%, 50%,25% or 10% of an HA dose. Unopsonized erythrocytes were

prepared by incubating the labelled cells with saline alone.Decreasing the concentration of antibody used to opsonizethe labelled erythrocytes resulted in decreased hepatic andincreased splenic uptake following i.v. injection, as well as

decreased total clearance by the spleen and liver combined(Fig. 1). The liver and spleen cleared less than 10% ofunopsonized erythrocytes, and less than 2% of the label was

in the lungs in any of the rats. The most specific splenicclearance occurred with erythrocytes opsonized with a 25%HA dose, which was therefore used for all subsequentexperiments.

Clearance in experimental ratsThe total erythrocytes cleared from the bloodstream by theliver and the spleen in the control rats was 71% (48-90%), thehemisplenectomized 71% (38-98%), the ties 50% (33-68%),the autotransplants 47% (35-60%), and the splenectomized47% (17-63%) (Fig. 2). The clearance was significantly greaterin the control and hemisplenectomized than in any of the other

243

M. T. R. Clayer et al.

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Fig. 2. The percentage of opsonized syngeneic erythrocytes removedfrom the circulation 3 h following i.v. injection into control rats (Con),or rats which had been hemisplenectomized (HS), splenectomized andautotransplanted (AT), splenectomized (SPX), or had the splenic arteryligated (Tie). Antibody-independent clearance was measured by inject-ing unopsonized erythrocytes into normal rats (saline).

groups (P < 0-02). There was no significant difference betweenany of the other three experimental groups.

The clearance of the erythrocytes by the liver and the spleenis shown in Fig. 3. The spleen in the controls phagocytosed25% (17-49%) of the injected erythrocytes, which was signifi-cantly more than in any of the other groups: 16% (4-50%) inthe hemisplenectomized, 3% (0 5-1 1%) in the ties, 2% (0O5-14%) in the autotransplants, and 2% (1-4%) in controlsinjected with unopsonized erythrocytes. Clearance by thehemisplenectomized spleens was significantly greater than byeither the tie or autotransplanted tissue (P < 0-05), but therewas no significant difference between the latter two.

The liver in the controls phagocytosed 38% (27-65%) oferythrocytes injected, compared with 49% (33-61%) in thehemisplenectomized, 47% (31-75%) in the ties, 50% (20-67%)in the autotransplants, 47% (25-63%) in the splenectomized,and 6% (5-8%) in controls injected with unopsonized erythro-cytes (Fig. 3). There was no significant difference between thegroups, except that hepatic uptake was significantly less in therats injected with unopsonized erythrocytes (P < 0-001).

To allow for differences in organ weight, the percentageuptake per gram of tissue was calculated (Fig. 4). For the splenictissue the uptake per gram was 22 3%/g (14-49%/g) in thecontrols, 19 2%/g (7 7-40 5%/g) in the hemisplenectomized,20 7%/g (2 7-35 5%/g) in the ties, 18 2%/g (0 9-55.2%/g) inthe autotransplants and 1-5%/g (0 8-5-1 %/g) in controls injected

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Fig. 3. The percentage of radiolabelled syngeneic erythrocytes cleared bythe liver (o) and spleen (e) following i.v. injection into control rats(Con), or rats which had been hemisplenectomized (HS), splenectomizedand autotransplanted (AT), splenectomized (SPX), or had the splenicartery ligated (Tie). Antibody-independent clearance was measured byinjecting unopsonized erythrocytes into normal rats (saline).

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Fig. 4. The percentage of radiolabelled syngeneic erythrocytes clearedper gram of tissue by the liver (o) and spleen (0) following i.v. injectioninto control rats (Con), or rats which had been hemisplenectomized(HS), splenectomized and autotransplanted (AT), splenectomized(SPX), or had the splenic artery ligated (Tie). Antibody-independentclearance was measured by injecting unopsonized erythrocytes intonormal rats (saline).

with unopsonized erythrocytes. There was no significant differ-ence in uptake per gram of splenic tissue between any of theexperimental groups. The uptake per gram of hepatic tissue didnot differ between any of the experimental groups.

Histological analysisThe autoradiographs of the spleens from control rats showedthat the label localized in the red pulp cords, with very littleuptake in the white pulp or marginal zone (Fig. 5).

The tie and autotransplanted splenic tissue had a markedreduction in the amount of white pulp and marginal zone. Thewhite pulp in the control spleen was 14-3% (11 -7-21 -6%) of thearea of the splenic section, compared with 15-6% (9 1-21 2%)in the hemisplenectomized, 1-6% (0 2-3 7%) in the tie and1l9% (0 1-4 5%) in autotransplanted splenic tissue. Thepercentage of marginal zone was similarly reduced. The per-

centage of red pulp was 70 4% (60 3-77 6%) in the controlsplenic tissue, 68-6% (54 8-79-4%) in hemisplenectomized,96-9% (91 1-99-8%) in the tie and 96-0% (87-8-99-9%) inthe autotransplanted.

The white pulp present in the regenerated tissue was almostalways at the periphery of the section, while the central regionconsisted mainly of red pulp cords and fibrous tissue. A centralarteriole was found in 89% (84-100%) of the white pulp areas

in the control spleens and 87% (79-96%) in the hemisplenec-

Fig. 5. The localization of phagocytosed radiolabelled erythrocytes in

normal splenic tissue demonstrated by autoradiography. The auto-

radiograph is on the top, with the corresponding photomicrograph ofthe haematoxylin and eosin-stained section below (x 12).

tomized, significantly more than the 36% (5-64%) in the ties or46% (11-64%) in the autotransplants (P < 0-001).

Efficiency of clearance by red pulpA relative index of the total amount of red pulp in the splenictissue was derived by multiplying the total weight of thesplenic tissue by the percentage area of the red pulp. Thecontrol spleens had a relative index of 76-5 (53-7-106-8),the hemisplenectomized 48-5 (32.9-288-7), the ties 21-3(8 3-37-5), and the autotransplants 11.1 (4 8-304). Therewas a significant correlation between this index of amount ofred pulp and the splenic uptake of the labelled erythrocytes(P < 0-001).

An esimtate of the uptake of erythrocytes per unit of redpulp was made by dividing the uptake per gram of splenic tissueby the percentage of red pulp as determined by the imageanalysis. The uptake per unit of red pulp in the controls was

31 5 (21-47), 27 (12-66) in the hemisplenectomized, 22 5(3-36) in the ties and 19 (1-58) in the autotransplants. Therewas significantly more uptake per unit of red pulp in thecontrols compared with the autotransplants (P < 0-005) or

the ties (P < 0-01), but no difference between the controlsand the hemisplenectomized.

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100 * functions in resistance to infection, the synthesis of specific° A opsonins or antibodies, and the clearance of poorly opsonized

* . 8 bacteria from the circulation. We have shown that antibody80 6O synthesis in humans and rats with regenerated splenic tissue is

impaired [18, 19], although significant and perhaps protectiveD3 *serum concentrations can be achieved by immunization. The

, 60 -' °purpose of this study was to test if the regenerated splenic tissueretained the ability to phagocytose poorly opsonized particles.

'40D Testing of the phagocytic function of the spleen has been-40 performed using heat-damaged erythrocytes, and found to be

significantly reduced in rats with splenic autotransplants [20].D20 This probably does not reflect phagocytosis, but the retention

of damaged erythrocytes which are less deformable thannormal, and physically trap in the splenic microvasculature

. | and are then removed. Experiments which measure the clear-o 60 120 180 ance of bacteria or survival following challenge with liveTime (min) organisms are difficult to interpret, because the outcome will

ig. 6. The percentage of the total radioactivity cleared from the blood depend not just on the phagocytic function of the splenic tissue,various times following the injection of radiolabelled opsonized but on the antibody present, the rate of bacterial replication,ngeneic erythrocytes into control rats (.), or rats which had been and the rate at which the animal synthesizes new antibody. Themisplenectomized (A), splenectomized and autotransplanted (J), amount and class of background opsonic antibody probably)lenectomized (o), or had the splenic artery ligated(m). varies between animals, and there is no method of ensuring that

the rate of bacterial replication is the same in each of theanimals. Protection by the spleen depends partly on the earlyand rapid production of opsonins for circulating infectious

ate of clearancefrom the blood agents, and this rate has been shown to vary between experi-13 control rats, eight hemisplenectomized, 10 auto- mental groups [21]. In this study phagocytic function was

ansplanted, 10 splenectomized and eight ties, the clearance assessed in a system in which the opsonization of the particlesom the bloodstream was measured at intermediate time could be controlled. Syngeneic erythrocytes, against which theDints as well as at 3 h. The amount cleared at the intermediate recipient rats had no antibody, were opsonized with the sameme points was expressed as a percentage of that cleared at amount of IgG antibody, and with an appropriate concen-30mi in each rat (Fig. 6). In each of the experimental groups tration, so that phagocytosis occurred preferentially in the

a significantly greater percentage of the label was cleared in thefirst 10min following injection, compared with the controls, butthere was no difference between the groups 60 or 120 min afterinjection.

DISCUSSION

This study has demonstrated that the splenic tissue whichregenerated following splenic artery ligation or splenic auto-transplantation in the rat was histologically and functionallyabnormal. The percentage of white pulp in these tissues wassignificantly reduced, with many of these white pulp areas nothaving a central arteriole. The percentage of red pulp, the zonein which phagocytosis of opsonized erythrocytes occurred, wascorrespondingly increased. The red pulp of the regeneratedsplenic tissue was less efficient in phagocytosis than controltissue, but because of the increased percentage of red pulp in theregenerated tissue, the clearance per gram did not differ fromcontrols. The regenerated splenic tissue was smaller than anormal intact spleen, and because of this reduction in size thetotal clearance of opsonized erythrocytes from the circulationin rats with regenerated splenic tissue was significantly less thanin controls, and not significantly different from that insplenectomized rats.

The regeneration of splenic tissue after autotransplantationor splenic artery ligation in patients and in animal models iswell described, but the capacity of such tissue to protect againstOPSI is controversial, some authors reporting benefit, othersno improvement [8, 9, 12-17]. The spleen has two major

spleen.It is unlikely that the altered clearance is a consequence of a

reduction in blood flow to the regenerated tissue. If the bloodflow were limiting, it would be expected that opsonizedparticles would be cleared from the circulation slower than ineusplenic rats, but the opposite occurred. The data suggest thatcapacity of the phagocytic organs to remove particles was

saturated more quickly in the experimental rats, with themajor difference between the experimental and control ratspresumably being the capacity of the spleen. This is supportedby the finding that the hemisplenectomized spleen, whichretained its normal blood supply, cleared just over half of theradiolabel of the control spleen. The impaired phagocytosismay result from the abnormal histology of the red pulp.Scanning electron microscopy of microvascular casts hasshown that the vascular channels in the red pulp of autotrans-plants are abnormal, with increased diameter and loss of thefine saccular dilatations found in normal spleens [22]. Thedecreased contact between the phagocytic cells which line thecords and the blood may contribute to the reduced efficiency ofclearance.

This study has confirmed that the spleen is the mostimportant organ for the removal from the bloodstream ofparticles which are poorly opsonized [2]. It also confirms thatthe lymphoid compartments of the spleen do not regeneratenormally in autotransplants [23-26] or ties [27], and suggestsfurther abnormalities in the vascular bed with the finding thatthe number of white pulp areas which have a central arteriolewere greatly reduced. As with radiolabelled colloid [25, 27], the

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IgG-mediated phagocytosis in regenerated splenic tissue 247

clearance from the blood and uptake by splenic tissue oferythrocytes with a low concentration of IgG was significantlyreduced in rats with regenerated splenic tissue.

The results reported here demonstrate that, in the rat,phagocytosis of opsonized erythrocytes occurred in the redpulp, and the red pulp of regenerated splenic tissue clearedopsonized erythrocytes less efficiently than control red pulp.Because of an increase in the percentage of red pulp in thetissue, the clearance per gram was not different from controls.However, because the regenerated tissue did not regrow to asufficient size, the clearance of opsonized particles from theblood was not different from splenectomized rats. Splenicautotransplantation would therefore only be expected to beof value in the maintenance of phagocytic function providedenough splenic tissue were restored. The factors which deter-mine this are not currently known.

ACKNOWLEDGMENTS

M.T.R.C. was in receipt of the Royal Australasian College of SurgeonsFoundation Kelly Memorial Scholarship. The support of the NationalHealth & Medical Research Council and the Royal Adelaide Hospital,and the skilled assistance of Mr J. Bellen, are gratefully acknowledged.

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