American Journal of Pathology, Vol 139, No. 4, October 1991Coprigbt C American Association of Pathologits

Mechanisms of Clearance of ImmuneComplexes from Peritubular Capillaries inthe Rat

Charles E. Alpers, Kelly L. Hudkins, PamPritzl, and Richard J. JohnsonFrom the Departments of Pathology and Medicine, School ofMedicine, University of Washington, Seattle, Washington

These experiments evaluated extraglomerular sites ofrenal immune complex (IC) deposition and specificfeatures ofhost capability to remove these IC. Ex vivoperfusion of rat kidneys with the endothelium bind-ing lectin concanavalin A (conA)followed by rabbitanti con A IgG results in a subendothelial IC nephri-tis in glomerular capillaries (GC) and diffuse ICfor-mation with complement (C3) deposition in peritu-bular capillaries (PC). Histologic, immunofluores-cence, and ultrastructural studies were performed at10 minutes and 1, 4, and 24 hours afterperfusion. At10 minutes, strong linear binding of con A, rabbitIgG, and rat C3 to the endothelium was detected byimmunofluorescence in both GC and PC In GC thiswasfollowed by endothelial cell swelling and denu-dation (1 hour) with platelet and neutrophil infil-tration andformation of subendothelial IC depositswhich persisted at 4 and 24 hours. In contrast, somePC endothelial swelling was also present at 10 min-utes and 1 hour, but ICs (IgG, con A, C3) were cappedand shed into capillary lumina at I to 2 hours withcomplete clearance of IC by 4 hours. Selective neu-trophil depletion, by antisera and irradiation, andcomplement depletion with cobra venom factor, de-layed clearance ofPC IC by several hours but com-plete clearance of IC with restored structural integ-rity of PC was still achieved by 24 hours. Plateletdepletion had no effect on PC IC clearance. Thesestudies demonstrate a modelfor study ofPC IC. Sucha model may aid our understanding oflupus nephri-tis in which extensive GCIC deposits associated withsevere inflammatory injury may coexist with PC de-posits. Efficient clearance ofIC in PC compared withGC may be due to differences in hemodynamicforces,amounts of IC formed in each of these sites, differ-ences in binding of IC to subendothelial basementmembrane, orphenotypic specialization of the endo-

thelium lining these two different capillary beds.(AmJ Pathol 1991, 139:855-867)

Extraglomerular renal capillary wall immune deposits inhumans are commonly encountered in biopsies obtainedfrom patients with proliferative lupus nephritis.1" Theyhave also uncommonly been reported in allograft kidneysundergoing rejection.1 7 The significance and natural his-tory of these deposits remains poorly understood, andour potential to achieve a better understanding remainshampered by the paucity of relevant experimental mod-els of this type of immune renal injury. This is particularlydisappointing in the context of the considerable recentadvances made in understanding of the role of the en-dothelial cell as both a target and modulator of immuneand inflammatory tissue injury.8'9We have approached this problem utilizing the con-

canavalin A (con A)/rabbit anti-con A antibody ex vivorenal perfusion model in the rat, which results in endo-thelial binding of the lectin con A, followed by subsequentformation of con A/rabbit anti-con A IgG immune com-plexes (IC) in situ on the endothelial and subendothelialsurfaces of the renal microvasculature.10'" Within theglomerulus, we have shown that subsequent glomerularinjury and proteinuria is dependent on the influx of neu-trophils and platelets as well as activation of comple-ment.' 112We have extended these studies to the renal peritu-

bular capillaries, where the renal artery perfusion of con Afollowed by antibody to con A also results in rapid forma-tion of IC in situ on the endothelial surface. We have foundthat there are considerable differences in the susceptibil-ity of the glomerular and peritubular capillary beds to per-sistence of the IC deposits and induction of inflammatoryinjury. Furthermore, mediators that contribute to capillarywall injury in glomerular capillaries, specifically white

Supported by grants DK40802, DK34198, and DK39068 from the Na-tional Institutes of Health.

Accepted for publication June 5, 1991.Address reprint requests to Dr. Charles E. Alpers, Department of

Pathology, RC-72, University of Washington MedicaJ Center, Seattle, WA98195.

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cells, and complement, are shown to instead have a ben-eficial role in peritubular capillaries in that they appear tofacilitate clearance of IC.

Methods

Description of Immune Complex Nephritis

Immune complex nephritis was induced in rats by theselective renal artery perfusion of the lectin concanavalinA (con A) followed by anti-concanavalin A antibody, asdescribed previously.11'13 The infusion of con A results inbinding to glucose and mannose residues on glomerularendothelial cells and basement membrane (GBM) glyco-protein, and hence allows it to function as a "planted"antigen.12 The subsequent perfusion of anti-con A anti-body results in formation of immune complexes in situ onthe endothelial cell surface.

Renal Artery Perfusion

The technique for inducing GN with con A and antibodyto con A has been described in detail previously.11Briefly, male Sprague-Dawley rats (Tyler Labs, Bellevue,Washington, USA) weighing between 200 and 300 gwere anesthetized with equithesin (pentobarbital andchloral hydrate) IP (0.3 cc per 100 g body wt), and a leftnephrectomy was performed. The aorta was isolatedabove and below the right renal and superior mesentericarteries, and blood flow was interrupted. The superiormesenteric artery was cannulated in a retrograde man-ner with a 30-gauge needle and the right kidney perfusedusing a constant infusion pump (Sage Instrument Div.,Orion Research Inc., Cambridge, MA) at a rate of 0.75ml/min on a heated operating table. The kidney was ini-tially perfused with 0.5 ml phosphate-buffered saline(PBS), pH 7.2 followed by 1.0 ml of PBS containing 125,ug con A (ICN Biomedicals, Costa Mesa, CA). After aflush of the cannula with 0.2 ml PBS, an additional infu-sion of polyclonal rabbit anti-con A IgG (19 mg in 0.5 mlPBS) followed by PBS (0.3 ml) was performed. The prep-aration of the anti-con A IgG has been described previ-ously.13 Blood flow was then restored to the kidney, theneedle removed, and bleeding from the puncture sitewas stopped by the application of gelfoam (Upjohn Co.,Kalamazoo, Michigan) and gentle pressure. Total isch-emia of the kidney was always less than eight minutes.The abdominal wound was then closed and the animalwas placed under a heat lamp for 1 to 2 hours until hehad recovered from anesthesia.

Experimental ProtocolThe rats used in this study were initially studied and re-ported in a separate investigation of the kinetics and leu-kocyte and platelet influx within the glomerulus in the conA/anti-con A model.12 Immune complex nephritis was in-duced in one group of rats by renal artery perfusion ofcon A and anti-con A antibody as described earlier (n =18). Control rats underwent renal artery perfusion withPBS alone (n = 16). Other groups studied included ratswith immune complex GN that had been previously de-pleted of complement (n = 16), leukocytes (n = 16), orplatelets (n = 16).

Experimental AnimalsEach group (n = 4) was killed at 10 minutes, 1 hour, 4hours, and 24 hours. At the time of sacrifice, the bloodwas sampled for white cell and platelet counts and C3levels, and renal tissue was obtained for histologic stud-ies. Two additional rats in the unmodified con A/anti-conA treated group were killed at 2 hours, for total n of 4, 4,2, 4, and 4 animals at 10 minutes, 1 hour, 2 hours, 4hours, and 24 hours, respectively in this group.

Complement (C) DepletionRats were depleted of complement with cobra venomfactor (CVF; Naja naja kaounthia, Diamedix Corporation,Miami, FL).14 Rats were injected with CVF 300 p1kg IP inthree divided doses beginning 24 hours before renal ar-tery perfusion and received an additional dose of 100p1kg IP on the day of surgery and induction of GN. Serialserum C3 concentrations were measured by radial im-munodiffusion15 to monitor the complement status of theanimals. Mean serum C3 concentrations on the day ofrenal artery perfusion and at the time of sacrifice were 4%of the baseline serum C3 level obtained before the firstinjection of CVF. CVF-treated rats exhibited a leukocyto-sis (peripheral white blood count 25,500 ± 3088/mm3 vs.14,132 ± 1059/mm2 in normal controls) but had normalperipheral platelet counts (756,000 ± 46,000 vs. 764,000± 44,000 in normal controls) at the time of disease induc-tion. 1 1

Leukocyte (WBC) Depletion

Rats were depleted of leukocytes (WBC) by irradiationwith shielding of the right kidney in a method modifiedfrom Tucker et al.16 After anesthesia with equithesin, ratswere placed on a rotating platform and the right kidney

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was shielded with a 3-cm thick lead block. Rats received800 total rads at a rate of 20 rads/min using a unidirec-tional 137Cs source. Dosimetry studies have confirmedthat the shielded kidney receives less than 10% of thetotal dose (that is, 60-70 rads). Three days after irradia-tion rats also were given 1.0 ml anti-PMN serum IV, thathas been described previously.13 On day 4 the renalartery perfusion of con A and anti-con A was performed.At the time of surgery all 16 rats had severe leukopenia(mean 80/mm3; normal controls 14,132 + 1059/mm3)and an absence of PMNs (0/mm3). The peripheral plate-let count was slightly depressed (556,000 ± 52,000 vs764,000 ± 44,000 in normal controls), but complementlevels were normal.11 In contrast, rats studied 24 hoursafter induction of GN (that is, day 5 postirradiation) wereleukopenic (mean WBC < 75/mm3) and thrombocy-topenic (pit ct 50,000 ± 8,000/mm3), at the time of sac-rifice.

Platelet Depletion

Rats were depleted of platelets with polyclonal goat an-tiplatelet IgG.1 1 A dose of 40 to 50 mg/i 00 g body weightIP results in a selective thrombocytopenia (mean plateletcount < 1 0,000/mm3) within 8 hours and is sustained forover 40 hours. Platelet depleted rats had a leukocytosis(white blood count 20,800 ± 2420/mm3 vs. 14,132 ±1059/mm3 in control animals) and a modest depressionin C3 (mean 61% ± 2% of normal) at the time of surgery.

Renal Morphology

Tissue for light microscopy was fixed in Sorenson's buff-ered formalin (10% formalin in 0.1 M Na phosphatebuffer, pH 7.2) for at least 4 hours, and was then dehy-drated in graded ethanols and embedded in glycometh-ylmethacrylate (Polyscience, Warrington, PA). Sections(2-3 ,um) were stained with periodic acid Schiff (PAS)reagent.

Tissue for immunofluorescence (IF) was embedded inOCT (Lab-Tech products, Miles, Naperville, IL), snap-frozen in isopentane, and sectioned with a Tissue-Tek 11Microtome/Cryostat (Miles).10 Con A was detected by in-direct immunofluorescence using a biotinylated goatanti-con A antibody (Vector Labs, Burlingame, CA) fol-lowed by fluorescein isothiocyanate (FITC)-conjugatedstrepavidin (Amersham). Rabbit anti-con A IgG was de-tected using FITC-conjugated goat anti-rabbit IgG, andrat C3 with FITC-conjugated goat anti-rat C3 (OrganonTeknika Corp., BCA Cappel, West Chester, PA). Sectionswere examined with a Leitz Ortholux 11 microscope with aPloempak 2.2 vertical fluorescence illuminator (E. Leitz

Inc., Rockleigh, NJ) and the intensity was graded semi-quantitatively from 0 to 4 + as described previously.11

Tissue for electron microscopy (EM) was fixed in half-strength Karnovsky's Solution (1% paraformaldehydeand 1.25% glutaraldehyde in 0.1 M Nacacodylate buffer,pH 7.0), postfixed in osmium tetroxide, dehydrated ingraded ethanols, and embedded in epoxy resin. Thinsections were stained with uranyl acetate and lead citrateand examined with a Philips 410 (Philips Export BV, Eind-hoven, The Netherlands) electron microscope.

Immune Complex Localization

To confirm the ingestion and removal of con A immunecomplexes by circulating blood cells, con A conjugatedto the electron dense label ferritin (Sigma, St. Louis) wasutilized for perfusion studies in unmodified rats sacrificedat 10 minutes, and 1 and 2 hours (n = 2 each group).Subsequent steps after con A-ferritin perfusion wereidentical to those described earlier. At sacrifice, tissuewas divided for renal morphologic studies as mentioned.Ferritin particles were localized by examination of micro-graphs obtained by transmission electron microscopy.

Results

Unmodified con Alanti-con AAntibody-mediated Injury

Glomerular Capillaries

The glomerular injury in this model has been previ-ously described,11'12 and is illustrated in Figure 1. Thedeposition of con A, rabbit anti-con A IgG and comple-ment on the glomerular endothelium, are each readilydetectable at 10 minutes after perfusion by immunofluo-rescence (Figure 2). There is at the same time rapid influxof platelets, which peaks within 10 minutes and subse-quent influx of neutrophils and mononuclear cells, withthe influx of neutrophils peaking at 1 to 4 hours after renalartery perfusion. The capillary wall localization of con A,rabbit IgG, and rat C3 noted at 10 minutes remains withinglomerular capillaries at similar intensity for the remainderof the 24-hour study period. Electron microscopy showedthe presence of an electron-dense "fuzzy" material (pre-sumed antigen-antibody deposits) that coated the endo-thelium at 10 minutes. By 1 hour, immune deposits re-main on the glomerular endothelial cell surface, but alsoare present as confluent subendothelial electron-densedeposits. The endothelium at this time is swollen and par-tially denuded.12 At 24 hours, the glomerular capillaryloops still contain numerous neutrophils, and in many

858 Alpers et alAJP October 1991, Vol 139, No. 4

Figure 1. Histologic appearance of renal parenchyma in unmodified animals at 1 hour (A) and 24 hours (B) after perfusion of conca-navalin A (con A) and rabbit-anti con A antisera. In (A), there is aprominent influx ofneutrophils in glomerular andperitubular capillaries,but little evidence of alteration of intrinsic renal structures. In (B), there is extensive glomerular injury with intracapillamy thrombosis andpersistence of neutrophils; tubules and peritubular capillaries are morphologically normal. A, B, X520.

capillary loops, there are intracapillary thrombi consistingof a mesh of degranulated platelets and fibrin. Endothe-lium frequently is denuded and there are diffuse, conflu-ent electron-dense deposits on the subendothelial as-pect of capillary basement membrane. Occasional small,discrete subepithelial deposits are also encountered inglomerular basement membranes at this time.

Peritubular Capillaries

In the peritubular capillaries, as in glomerular capillar-ies, at 10 minutes after perfusion of con A and anti-con A,there is intense (4 +) linear immunofluorescence stainingshowing the presence of con A, rabbit anti-con A IgGand rat C3 (Figure 2). Although there is no evidence ofperitubular capillary injury by light microscopic study,electron microscopy shows that there is a diffuse coat ofelectron-dense "fuzzy" material on the luminal surface ofthe peritubular capillary endothelium similar to that seenin glomerular capillaries associated with focal endothelialcell swelling. As in the glomeruli, there is also a prominentinflux of platelets into these peritubular capillaries; thisinflux is maximal between 10 minutes and 1 hour afterperfusion (Figure 3). By 1 hour, the intense immunofluo-rescence staining for con A, rabbit IgG, and C3 persist,but already there is evidence of focal capping and ag-gregation of immune complexes as indicated by a trans-formation from a "linear" to an increasingly granular pat-tern of staining along the peritubular capillary endothelial

surface (Figure 2B, 2C). Ultrastructural examination atthis time shows there is an influx of neutrophils, whichappear to be phagocytosing all residual immune depos-its as well as any associated endothelial cytoplasmic de-bris (Figure 4).

By 2 hours, the transition to a granular and less in-tense (2+) pattern of immunofluorescence staining inperitubular capillaries for each of con A, rabbit IgG, andC3 is well established, and it appears numerous immunecomplexes are being shed into the capillary lumina (Fig-ure 2). Ultrastructural examination shows persistence ofthe intraluminal neutrophils. At this time much of the elec-tron-dense material coating endothelial cells has disap-peared, a process associated with restoration of endo-thelial cell integrity. By 4 hours, the immune injury in theglomerulus persists, as indicated earlier, while peritubu-lar capillary involvement has completely resolved withcomplete disappearance of con A, rabbit IgG, and com-plement from the endothelial cell surface as detected byboth immunofluorescence and electron microscopy.

Studies Utilizing a Ferritin Label to con A

Studies in unmodified animals in which con A wasconjugated to the electron-dense label ferritin but other-wise following exactly the experimental protocol de-scribed earlier showed similar pattems of injury as iden-tified by histologic and immunofluorescence examina-tion. Ultrastructural study localized the ferritin-labeled

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Figure 2. Composite oftypical immunofluorescencefindingsfor localization ofrabbit IgG (anti-con A antibody) in unmodified animals at10 minutes (A), 1 hour (B), 2 hours (C) and 4 hours (D) after perfusion ofcon A and anti-con A. The intense staining ofglomerular andperitubular capilklaies seen in (A) persists at 1 hour, but there is evidence offocal capping and aggregation of immune complexes asindicated by transformation from a "linear" to an increasingly granular pattern of staining along the peritubular capillaries endothelialsurface (arrows). At 2 hours, this granularpattern in peritubular capillaries is well established, and it appears complexes are being shed intothe capillary lumina. At 4 hours the immune injury in the glomerulus persists, while peritubular capillary involvement has resolved.Localization of con A and rat C3 shows a similar pattern. All, X365.

material to the electron-dense "fuzzy" material coatingthe endothelium in both glomerular and peritubular cap-illaries. Influxing platelets in both capillary beds showedfeatures of activation including centralization of granulesand/or degranulation, and filopodia formation. In addition,ferritin-labeled material identical to that coating the endo-thelium could be readily identified within vacuoles and/orcisternae in a minority of the platelets present, suggestiveof a process of either endocytosis or phagocytosis (Fig-ure 3). Clear evidence of ingestion and phagocytosis offerritin-labeled material by neutrophils was seen at 1 and2 hours after perfusion (Figure 4). In unmodified animalsthe aggregation and phagocytic activities of the neutro-phils coincided with the almost complete disposal of de-posits as determined by ultrastructural visualization.

Effect of Complement Depletion onPeritubular Capillary Injury

At 10 minutes, immunofluorescence studies for con Aand rabbit IgG in this group of rats showed similar exten-

sive, predominantly linear, immune complex formation inperitubular capillaries similar to that seen in the unmodi-fied animals (Figure 5). At 1 hour, this group continued toshow extensive immune complex deposition in peritubu-lar capillary walls with linear staining (Figure 6). Morpho-logic studies showed that the influx of platelets and neu-trophils into capillary lumina was largely abolished bycomplement depletion. At 4 hours, there was delayedresolution of peritubular capillary immune complexes ascompared with unmodified rats with IC nephritis (Figure7). At this time point, there was evidence of a process ofcapping with numerous immune complexes still presenton the endothelial cell surface of peritubular capillaries asdetected by immunofluorescence microscopy. However,by 24 hours, there was complete clearance of the peri-tubular capillary immune complexes as detected by im-munofluorescence microscopy, similar to what was seenat 4 hours in control rats with nephritis (Figure 8). Immu-nofluorescence stains for C3 were appropriately negativeat all times.

At each time point throughout the 24-hour period, ex-tensive, predominately linear, immune-complex deposi-

860 Alpers et alAJP October 1991, Vol. 139, No. 4

Figure 3. Electron micrograph shouwng influxofplatelets inperitubular capillary 10 minutesafter perfusion of con A and anti-con A IgG.The endothelium is coated by ill-defined'fuzy" material, some of which also appearsto bepresent in platelet vacuoles and/or cister-nae. x26,250.

tion, which was indistinguishable from the glomerular de-posits seen in unmodified animals (Figures 5-8), per-sisted in glomerular capillaries.

Effect of White Blood Cell Depletion onPeritubular Capillary Immune-complexInjury

Severe leukocyte depletion (mean peripheral WBC count80/mm3) was accomplished in 16 rats with a combination

of irradiation and anti-PMN serum before induction of ne-phritis. As in complement-depleted rats, there was strongstaining by immunofluorescence microscopy for con Aantigen, rabbit IgG, and rat C3 at 10 minutes in bothglomerular and peritubular capillaries; this staining wasindistinguishable from unmodified rats with immune-complex nephritis (Figure 5). At 1 hour, immunofluores-cence studies in this group were similar to that in com-plement-depleted group in that there was extensive im-mune-complex deposition in capillary walls (Figure 6).Leukocyte depletion had no effect of the initial platelet

Figure 4. Electron micrograph showing aneutrophilphagocytosing residual con A/anti-con A immune complexesfrom the endotheli-al suface at 1 hour. The con A has been con-jugated to the electron dense label ferritin.Some immune complexes are already presentintracellularly in a phagolysozome (arrow).Large portions of the capillary endotheliumvisualized in this micrograph already showcomplete shedding of complexes and anormal morphologic appearance includingthe presence of fenestrae (small arrow).X13,100.

Renal Peritubular Capillary Immune Injury 861AJP October 1991, Vol. 139, No. 4

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Figure 5. Composite of typical immunofluorescence localization ofrabbit IgG (anti-con A antibody) at 10 minutes in (A) unmodified, (B)platelet depleted, (C) neutrophil depleted, and (D) complement depleted groups. All groups show extensive immune complex deposition inglomerular (shown in A only) andperitubular capilklry walls. Localization ofcon A and rat C3 showed a similar pattern in groups (A)(B)and (C) C3 was not detectable in (D). All, x365.

accumulation seen at 10 minutes and 1 hour. At 4 hours,there was again evidence of marked delay in clearanceof peritubular immune deposits as detected by immuno-fluorescence when compared with unmodified rats withimmune-complex nephritis (Figure 7). As in complement-depleted rats, there was evidence of extensive cappingof immune-complex aggregates on the endothelial cellsurface; these aggregates showed complete clearanceat 24 hours (Figure 8).

Effect of Platelet Depletion on PeritubularCapillary Immune Complex Nephritis

Severe thrombocytopenia (mean peripheral plateletcount < 1 0,000/mm3) had no effect on the extent of de-position of con A antigen, rabbit IgG, or rat C3 in eitherglomeruli or peritubular capillaries as detected by immu-nofluorescence microscopy when compared with normalunmodified rats with immune-complex nephritis at any ofthe time points examined (Figures 5-8). Platelet depletionhad no effect on recruitment of neutrophils into peritubu-lar capillaries at 1 hour. No discernible changes in eitherendothelial cell morphology or extent of electron-dense

"fuzzy" material on the endothelial cell surface was de-tectable by ultrastructural studies in this group.

Discussion

These studies define a process of antibody-mediated re-nal microvascular injury whereby antibodies directed toan antigen planted on the surface of peritubular capillaryendothelium form immune complexes in situ, which un-der normal conditions are then efficiently removed within4 hours. Within this time frame, the IC remain confined tothe endothelial cell surface and are not detected in per-itubular capillary or tubular basement membranes. Ourmorphologic studies show this process of immune-complex formation is associated with activation and de-position of complement, early intravascular recruitment,and activation of platelets, and subsequent recruitment ofneutrophils. Although it is known that each of these me-diators may play a role in clearance of immune com-plexes, the clearance of the con A/anti-con A immunecomplexes from the peritubular capillary endothelial sur-face seems to involve primarily a process of clusteringand shedding analogous to that previously described in

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Figure 6. Composite of typical immunofluorescence localization of rabbit IgG (anti-con A antibody) at 1 hour in (A) unmodified, (B)platelet depleted, (C) neutrophil depleted, and (D) complement depleted groups. All groups continue to show extensive immune complexdeposition in glomerular andperitubular capillary walls. Localization ofcon A and rat C3 showed a similarpattern in groups (A)(B) and(C) C3 was not detectable in (D). All, x365.

studies of ligand binding to lymphocyte surface anti-6,17,18 inaniotesingens, and in studies of anti- angiotensin-

converting enzyme antibody-mediated injury to lung andglomerular endothelium.19,20 These studies have shownthat because of the fluid mosaic structure of cell mem-branes, a ligand, in the present case anti-con A antibody,possibly can bind to structures normally present orplanted in the plasma membrane with subsequent redis-tribution of these bound complexes into discrete aggre-gates on the cell surface ("capping"). These aggregatesare cleared from the cell surface either by endocytosis, oras in this model, by shedding from the cell surface intothe vascular space. This process of capping and shed-ding of IC may be a special feature of microvascularendothelium or the antigen/antibody system used; othershave had difficulty demonstrating this process in glomer-ular epithelial cells in vitro using different antigen/antibody systems with alternate, principally electrostatic,mechanisms of ligand binding.21

The effect of the depletion experiments involving com-plement, neutrophils, and platelets showed only limitedmodification of IC clearance and resolution in peritubularcapillary injury, in contrast to our previous studies inwhich identical manipulations dramatically altered the

natural evolution of glomerular capillary injury.11 12 In theglomerulus, it has been shown that each of these com-ponents plays an essential role in augmenting inflamma-tory injury and glomerular dysfunction. The specificmechanisms by which neutrophils, complement, andplatelets interact in the process of resolution of immune-complex deposition in the peritubular capillaries is notwell understood. The early influx of platelets, with addi-tional features of platelet activation and extensive contactwith capillary endothelium as well as the presence ofelectron-dense material within platelet vacuolar struc-tures and/or cisternae, have suggested that in the rat,platelets are able to endocytose, and possibly phagocy-tize, immune complexes. It follows that platelets may thushave a role in antigen processing and/or presentation, orrecruitment of other inflammatory cells. To further estab-lish this possibility, several rats were perfused with con Aconjugated to the electron-dense label ferritin, which wasfollowed by anti-con A antibody following the protocoldescribed earlier. These rats showed accumulation offerritin-bound electron-dense material, presumably rep-resenting con A/anti-con A immune complexes, withinplatelets at 10 minutes. Others have also observed ap-parent platelet phagocytosis in experimental and in vitro

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Figure 7. Composite of typical immunofluorescence localization of rabbit IgG at 4 bours in (A) unmodified, (B) platelet depleted, (C)neutropbil depleted, and (D) complement depleted groups. Control andplatelet depleted animals have near complete resolution ofperitu-bular capillavy, but not glomerular deposits, as seen in (A) and (B); neutrophil and complement depleted rats show delayed resolution ofthe peritubular capillary immune complexes. Localization ofcon A and rat C3 showed a similar pattern in groups (A)(B) and (C) C3 wasnot detectable in (D). All, X365.

settings.2244 Nonetheless, it is our impression based onobservations of transmission electron micrographs and inthe absence of any quantifiable data, that the ingestionand possibly phagocytic activities of the platelet exhib-ited in our studies is likely to involve a minor portion of theimmune-complex disposal in unmodified animals com-pared with the readily identified, extensive phagocytosisof deposits by neutrophils. It is clear from our ablationstudies that the platelets have no essential role in theclearance of immune complexes from peritubular capil-lary endothelium in this model.

Complement might participate in the removal of im-mune complexes via a number of mechanisms. In addi-tion to its chemoattractant effect for inflammatory andphagocytic cells, complement has a well-known role inimmune-complex removal through the process of op-sonization.25'26 An additional role for complement in sol-ubilizing immune complexes to aid in their removalthrough the circulation has been recently reviewed.2627The delayed clearance of peritubular capillary deposits incomplement-depleted animals may be due to one or allof these mechanisms. However, the finding that the im-mune complexes can be removed within 24 hours in

complement-depleted animals is indicative that none ofthese processes are essential for immune-complexclearance. Similarly, although neutrophil phagocytosis isreadily identified in control animals, the fact that clear-ance of deposits is delayed but not prevented by neu-trophil depletion indicates that the neutrophil is helpful butnot required for the endothelial cell to respond effectivelyto this form of immune-complex injury.

A striking finding of this study is the divergence inresponse to an identical form of immune-complex-mediated injury by the two principal capillary beds of thekidney. The glomerular injury in this model has been pre-viously described and illustrated (Figure 1; 11.12). Afterdeposition of con A, rabbit anti-con A IgG and comple-ment on the glomerular and peritubular capillary endo-thelium, there is a rapid influx of platelets within 10 min-utes and subsequent influx of neutrophils and mononu-clear cells. However, within glomeruli the immunecomplexes are not cleared by capping and shedding,and there is an evolution to a severe proliferative glomer-ulonephritis with prominent intracapillary thrombus for-mation and occasional segmental necrosis of capillaryloops, as seen in Figure 1. Ultrastructural examination of

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Figure 8. Composite of typical immunofluorescence localization of rabbit IgG at 24 bours in (A) unmodified, (B) platelet depleted, (C)neutrophil depleted, and (D) complement depleted groups. All groups are similar in showing persistent glomerular deposits with virtuallycomplete clearance ofimmune complexesfrom peritubular capillaries. Localization ofcon A and rat C3 showed a similarpattern in groups(A)(B) and (C) C3 was not detectable in (D). All, X365.

glomeruli at 24 hours after initiation of injury shows exten-sive denudation of capillary endothelium, extensive con-fluent subendothelial electron-dense deposits, and infre-quent, small subepithelial-dense deposits. This glomeru-lar process develops while adjacent peritubularcapillaries show efficient clearance of immune com-plexes and complete restoration of endothelial cell integ-rity.

How can we explain these dramatic differences ofcomplete resolution versus severe persistent injury in ad-jacent capillary beds? By both immunofluorescence andultrastructural studies, despite a first passage through theglomerular capillary beds, enough con A and anti-con Aantibody remain in the renal circulation to allow bindingover the surface of virtually the entire peritubular capillarybed. There they form immune complexes, activate com-plement, and attract platelets and neutrophils, much as inthe glomerular capillaries. However, we have no methodat present that would allow us to quantitate the amount ofcon A and IgG deposits in peritubular capillaries. There-fore we cannot directly address the critical question ofwhether the observed differences in the morphologic in-jury might be attributable to lesser quantities of either an-

tigen or antibody in the peritubular deposits. It thereforeremains a plausible hypothesis that lesser amounts of ICformation within the peritubular capillary bed result in lessinjury to the endothelial lining compared with the glomer-ular capillaries. With lesser degrees of injury, the endo-thelial cell may be either better able to handle IC utilizingthe capping and shedding responses described in thisstudy or may present a stronger barrier to IC access tothe underlying basement membranes where such com-plexes might bind in a manner that precludes rapid dis-posal.

An alternate basis for the difference in morphologicinjury may be the result of differences in hemodynamicforces and hydraulic conductivity between the glomeru-lar and peritubular capillary beds, with a higher glomer-ular intracapillary pressure serving to "drive" the immunedeposits into the capillary wall and/or somehow precludethe endothelial cell from mounting an effective clearanceresponse. The studies of Matsuo, et al., which utilized analternate endothelial-binding lectin (Helix pomatia) andanti-lectin antibody to induce glomerulonephritis alsobear on this possibility.28 In that model, the glomerularendothelium was preserved, but complexes were not

Renal Peritubular Capillary Immune Injury 865AJP October 1991, Vol. 139, No. 4

capped and shed. Rather, IC were found to migrate intothe glomerular capillary wall. The authors believed thisresult could be attributable to either hemodynamic forceswithin the glomerular capillary, or alternatively to compro-mise of the endothelial barrier to IC access to the glomer-ular basement membrane because of local inflammationor to initial formation of IC in the subendothelial spacewith subsequent binding to the glomerular basementmembrane. No evidence of peritubular capillary IC for-mation can be deduced from the immunofluorescencemicrographs in that article, and so direct comparisons tothe present study are limited. Nonetheless, in the ab-sence of a methodology that would allow us to measurehemodynamic forces or hydraulic conductivity in situ inthe peritubular capillary bed, the hypothesis that differ-ences in these parameters in the two capillary beds ac-counts for the differences in handling IC cannot yet beevaluated.

A third explanation for the differences in disease ex-pression may be derived from differences in the biologyof the endothelial cells lining these two capillary beds.Although intriguing, little is known about any such differ-ences. Both the glomerular and peritubular capillaries arelined by fenestrated endothelium, as seen in Figure 4,although the fenestrae of the peritubular microvascula-ture may not be as evenly distributed and hence asreadily identifiable as that of the glomeruli.2'31 Immuno-histochemical and enzyme histochemical studies haveshown similar expression of MHC class and 11 antigens,and similar capacities to bind such endothelial markersas Ulex europaeus lectin32 by these two capillary beds.Some differences have been identified, including thepresence of the enzymes alkaline phosphatase and AT-Pase in normal peritubular capillary but not glomerularendothelium.33 The significance of each of these findingsis unknown, but would support a premise that heteroge-neity of renal capillary endothelium could contribute tothe observed differences in capacity to respond to spe-cific, defined injury.

Although our studies clearly document a dissociationbetween glomerular and peritubular capillary injury withinthe same animal, our findings in peritubular capillariesbear noteworthy resemblance to findings by Fries et al.,in their studies of subendothelial immune complexes inrat glomeruli.3 In that model of con A/anti-con A medi-ated injury, in which there are several important differ-ences in the experimental protocol compared with themodel described herein, the investigators described theinitiation and evolution of glomerular injury over 24 hoursthat parallels to a great degree the course of unmodifiedperitubular capillary injury. That study showed immune-complex formation in situ was followed by complementdeposition, influx of platelets followed by neutrophils, ap-

parent shedding of complexes into the vascular space,and complete resolution of the injury over a longer timecourse but which was complete by 24 hours. For reasonsnot entirely clear, the differences in experimental protocolbetween our study and that of Fries et al., result in dra-matically attenuated glomerular endothelial injury in theirmodel, in which the processes of glomerular endothelialdenudation, persistence of immune complexes, and de-velopment of functional impairment of the glomerularcapillary wall as manifest by proteinuria are not achieved.Injury to peritubular capillaries in their study was not de-tailed. Nevertheless, their studies do document that un-der some circumstances glomerular endothelium is ca-pable of responding to immune complex deposition in amanner similar to peritubular capillary endothelium, andso lend support to those hypotheses that implicate he-modynamic forces or local concentrations of antigen andantibody as the principal explanation for the differencesobserved in our study.

These studies may enhance our understanding of hu-man immune-mediated tubulointerstitial disease. Endo-thelial cell clearance may be an effective mechanism inprotecting the kidney parenchyma from some forms ofantibody-mediated injury, accounting for the infrequentoccurrence of such forms of injury within the tubulointer-stitial compartments with the important exception of dif-fuse proliferative lupus nephritis noted earlier.1- The per-itubular capillary bed may be much less successful indefending against cell-mediated forms of immune injury,as suggested by studies of experimental and human re-nal transplant rejection and other forms of interstitial ne-phritis such as drug-allergy-associated hypersensitivityreactions.'7 Finally, the development of models of per-itubular capillary injury may be of particular utility in viewof the observations by Bohle et al., whose studies of hu-man glomerulonephritis have led to the premise that in-jury and obliteration of the peritubular capillary bed is akey element in the development of interstitial fibrosis,which in turn has been recognized as the critical param-eter in establishing permanent renal impairment even inpatients with a primary glomerular injury.8

In summary, this study defines the initiation and evo-lution of a specific antibody-mediated form of endothelialinjury in the renal peritubular vascular bed, and has fur-ther characterized how the response to injury may bemodified when each of three important arms of the in-flammatory response are depleted. These studies docu-ment a remarkable capacity for some endothelial cells todefend themselves against immune injury. The basis forthe dissociation between extent and resolution of glomer-ular and peritubular capillary injury observed in this studyremains to be established. It does seem likely that theintegrity of the endothelial cell, both as a barrier govern-

866 Alpers et alAJP October 1991, Vol. 139, No. 4

ing accessibility of antigen and antibody to underlyingbasement membranes and as an active participant in thehandling of immune complexes, is an important determi-nant of the extent of microvascular injury engendered bythe con A/anti-con A model.

Acknowledgment

The authors thank Tamara Carlson for her expert secretarial as-sistance.

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