Kidney International, Vol. 45 (1994), pp. 32—42

ICAM-1 directs migration and localization of interstitialleukocytes in experimental glomerulonephritis

PRUDENCE A. HILL, Hui Y. LAN, DAVID J. NIKOLIC-PATERSON, and ROBERT C. ATKINS

Department of Anatomy, The University of Melbourne, Parkville, and Department of Nephrology, Monash Medical Centre, Clayton,Victoria, Australia

ICAM-l directs migration and localization of interstitial leukocytes inexperimental glomerulonephritis. Recent studies of rat anti-GBM dis-ease have demonstrated a functional role of the ICAM-1ILFA-1 inter-action in the entry of leukocytes into the glomerulus and an associationbetween interstitial ICAM-1 expression, leukocyte infiltration andtubulointerstitial damage. In the current study, we used immunogoldultrastructural techniques to identify ICAM-l/LFA-l interactions in theinitiation of interstitial leukocyte infiltration during the first 24 hours ofrat accelerated anti-GBM disease. In normal rats, there was weakconstitutive ICAM-1 expression in the interstitium: on the endothelialluminal surface of interstitial capillaries, venules and arterioles, on theentire surface of interstitial fibroblast-like cells and confined to thebrush border of proximal tubules. As early as 1.5 hours after injectionof anti-GBM serum, there was a marked increase in the intensity ofICAM-1 expression, most notably on capillary endothelium, fibroblast-like cells and brush borders of proximal tubules, particularly in theperiglomerular/perihilar areas. Mononuclear leukocytes exhibitingstrong surface LFA-l (CD1 la and CD18) expression were seen adher-ent to the endothelium of interstitial capillaries, with ICAM-1 andLFA-l antigens present at sites of contact. In addition, mononuclearcells migrating into the interstitium showed areas of close apposition tointerstitial fibroblast-like cells, and here ICAM-l and LFA-1 expressionwere also prominent at the sites of contact. This is the first study todemonstrate sites of ICAM.1/LFA-l interaction in mononuclear cellmigration and localization in glomerulonephritis. The results suggestthat up-regulation of periglomerular/peritubular capillary ICAM-1 ex-pression is important for mononuclear cell entry into the interstitium,while interaction with fibroblast-like cells may facilitate movement andsubsequent focal accumulation of mononuclear cells at sites within theinterstitium.

It is now well-recognized that most forms of glomerulone-phritis have a significant interstitial mononuclear leukocyticinfiltrate which correlates better with the degree of renalinsufficiency than does the severity of the glomerular lesion[1—5]. In experimental accelerated anti-glomerular basementmembrane glomerulonephntis (anti—GBM ON) interstitial leu-kocytic infiltration becomes evident within 12 hours, initially ina perivascular sheath surrounding glomerular hilar arteriolesand then becoming widespread to periglomerular and tubuloin-terstitial areas [2].

It is now apparent that leukocyte entry into tissues is con-

Received for publication May 13, 1993and in revised form July 27, 1993Accepted for publication July 29, 1993

C 1994 by the International Society of Nephrology

32

trolled by the dynamic interaction between adhesion moleculesexpressed by leukocytes and the endothelium [6—9]. The inter-cellular adhesion molecule-l (ICAM-1, CD54), a cell surfaceglycoprotein belonging to the immunoglobulin superfamily, isexpressed on many cell types including endothelium [10] and isup-regulated by proinflammatory cytokines (IL-i, TNF and yIFN) [6, ii]. ICAM-1 plays a major role in T lymphocyte andmacrophage adhesion to activated endothelium via binding tothe ligand lymphocyte function-associated antigen-I (LFA-i)[6, 8, 11—14]. LFA-1, a member of the (32 integrin family, ismade up of an a chain (CD1 la) and a /3 chain (CD18) and isexpressed on most leukocytes, particularly activated T cells [7,151.

The functional importance of the ICAM-l/LFA-i interactionin the leukocytic accumulation in rat anti-GBM GN has recentlybeen demonstrated by in vivo antibody blocking studies [16].Furthermore, treatment of rat anti-GBM ON with the IL-ireceptor antagonist has demonstrated that up-regulation ofinterstitial ICAM-l expression is associated with interstitialleukocytic infiltration and tubulointerstitial damage (Nikolic-Paterson et al, manuscript submitted).

A number of light microscopy studies have examinedICAM-1 expression in renal diseases [17—26], but the exactlocalization of ICAM-i in glomerulonephritis remains to beestablished. Therefore the aim of this study was to determinethe precise ultrastructural localization of ICAM- 1 expressionand ICAM-i/LFA-l interactions during the initiation of theinterstitial leukocytic infiltration in anti-GBM ON in the rat.

Methods

Animals

Inbred male Sprague-Dawley (SD) rats aged three to fourmonths were obtained from the Monash Medical Centre AnimalHouse.

Experimental anti-GBM glomerulonephritis

Rabbit nephrotoxic serum (NTS) was raised as previouslydescribed [21. Passive accelerated anti-GBM GN was inducedin SD rats by subcutaneous immunization with 5 mg of normalrabbit IgG (Silenus, Australia) in Freund's adjuvant followedfive days later by intravenous injection of NTS (10 mtfkg bodywt). Groups of two animals were sacrificed at 1.5, 6, 12 and 24hours. A group of normal animals was used as controls.

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Monoclonal antibodiesMouse monoclonal antibodies used in this study were as

follows: 1A29 [27], an IgGl to rat ICAM-l (CD54); WT1[28], anIgG2a to rat LFA-l a chain (CD! la); WT3 [28], an IgG 1 to ratLFA-l/3 chain (CD18). All antibodies were supplied by Serotec(Australian Laboratory Services, Melbourne, Australia).

ImmunohistochemistryKidney for immunoperoxidase staining was immersion fixed

in periodate-lysine-paraformaldehyde (PLP) for two hours.Serial 6 js cryostat sections were cut and stained as previouslydescribed [2]. The mouse monoclonal antibodies were visual-ized with a three layer immunoperoxidase technique usingperoxidase-conjugated goat anti-mouse IgG and mouse peroxi-dase anti-peroxidase complexes followed by development with3'3'-diaminobenzidine (DAB, Sigma Chemical Co., St. Louis,Missouri, USA) and counterstaining with Harris' hemotoxylin.

ImmunoelectronmicroscopyThe kidneys were perfusion fixed with PLP for five minutes

and then 1 mm thick slices were immersion fixed in PLP for twohours. The slices were then immersed in increasing concentra-tions of sucrose (5% up to 20%) in 0.1 M phosphate bufferedsaline (PBS) pH 7.4 over 36 hours, and then placed in OCT andsnap frozen in liquid nitrogen. Subsequently 30 i cryostatsections were cut and incubated in 5% normal goat serum(NGS) in 0.01 M PBS, 0.8% bovine serum albumin (BSA) and0.1% IGGS quality gelatin as a blocking step and then in theprimary monoclonal antibody overnight at 4°C. Monoclonalantibodies were diluted 1 in 75 in 1% NGS in 0.01 M PBS/O.8%BSA/0. 1% gelatin (dilution buffer). For each animal controlsections were incubated in dilution buffer only. Sections werethen incubated in goat antimouse IgG labeled with 1 nm goldparticles (Auroprobe One, Amersham Industries, ArlingtonHeights, Illinois, USA) diluted 1 in 25 in dilution buffer.Following extensive washing sections were fixed in 2% glutar-aldehyde in PBS for 15 minutes and then silver enhanced usingIntenSE M kit (Amersham). Post-fixation with 1% aqueousosmium for 30 minutes was followed by dehydration in gradedacetones and flat embedding of the sections in Epon-Araldite.Sections 0.08 s thick were cut straight from the surface of theblock, mounted on copper grids, stained with lead citrate anduranyl acetate and examined in a Philips CM12 electron micro-scope.

Results

ImmunohistochemistryIn normal animals ICAM- 1 (CD54) was constitutively weakly

expressed in glomeruli, in the interstitium and on some proxi-Fig. 1. (a) ICAM-1 (CD54) in normal. ICAM-1 is weakly expressed inmal tubule brush borders (Fig. la). The tubulointerstitial the renal cortical interstitium, proximal tubule (PT) brush borders and

ICAM-1 expression was increased three- to fourfold as early as glomeruli (G). (b) ICAM-1 (CD54) in anti-GBM GN at 12 hours.1.5 hours after initiation of anti-GBM GN and maintained at ICAM-1 expression is markedly increased as compared with normal insimilar intensity throughout the 24-hour time period. In addition the periglomerular interstitium (arrows) and on proximal tubule (PT)

brush borders. 0, glomerulus. (c) LFA-1/3 (CD18) in anti-GBM GN atttie percentage Oi i.vt-t positive tuoujes rncreaseu. tiiiS 12 hours. This plate represents a serial section to that stained withup-regulation in tubulointerstitial ICAM-1 expression was more ICAM-1 in (b). The sites of CD18 positive leukocytes within themarked in the periglomerular/perihilar regions and adjacent interstitium (arrows) correlate with those areas of most intense inter-peritubular areas (Fig. ib). There was an associated interstitial stitial ICAM-l expression in (b). G, glomerulus. x200.infiltration of LFA- 1 (CD1 la/CD 18) positive leukocytes in thesame areas as the increased ICAM-l expression as demon-

34 Hill et a!: ICAM-1 in anti-GBM GN

Fig. 2. ICAM-1 (CD54) in anti-GBM GN at 6 hours. ICAM-l is present on the luminal surface only of a fenestrated interstitial capillary (arrows).Note ICAM-l expression on the surface of an interstitial fibroblast-like cell (F). ICAM-l is absent from the basal surface of an adjacent proximalconvoluted tubule (PCT). C, capillary lumen. x 18,000.

strated in serial sections (Fig. 1 b and c), however, preciselocalization of ICAM-l expression and sites of ICAM-l/LFA-linteraction could not be identified.

ImmunoelectronmicroscopyNormal animals. Within the cortical tubulointerstitium,

staining for ICAM-1 (CD54) was present on the luminal surfaceof the fenestrated endothelium of interstitial capillaries and theendothelium of venules and arterioles and on the whole surfaceof fibroblast-like interstitial cells. These fibroblast-like cellswere characterized by multiple long attenuated cell processesand prominent profiles of granular endoplasmic reticulum andGolgi apparatus. Sparse ICAM-l staining was detected on thefree surface of the parietal epithelium lining Bowman's capsule.ICAM-l was sparsely present on proximal tubular brush bor-ders but no staining was present on the lateral or basal surfacesof these cells. Tubular cells of the distal nephron were negative.Control sections were negative.

Occasional interstitial macrophages expressed surface LFA-l(CDI la and CD18). Control sections were negative.

Anti-GBM disease. As early as 1.5 hours mononuclear leu-kocytes, both macrophages and lymphocytes, were seen adher-ent to capillary endothelium and had migrated into the intersti-tium. This initial infiltrate was most prominent in the

periglomerular/perihilar regions. Subsequently mononuclear leu-kocytic infiltration became more widespread over 6, 12 and 24hours but was still most intense adjacent to glomeruli.

An increase in ICAM- 1 (CD54) staining on the endothelialluminal surface of interstitial capillaries was apparent at 1.5hours compared to normal and was further progressively in-creased up to 24 hours; this was most marked in the periglom-erular/perihilar regions (Fig. 2). Endothelial ICAM-1 expressionwas particularly prominent at the sites of adhesion of "active-appearing" mononuclear leukocytes (Fig. 3). These "active-appearing" leukocytes were characterized by abundant cyto-plasm with prominent Golgi apparatus, rough endoplasmicreticulum and electron dense lysosomal-type granules. Adher-ent leukocytes also expressed sparse surface ICAM-1 (Fig. 3).

In this disease model there was also up-regulation of ICAM- 1expression by interstitial fibroblast-like cells compared to nor-mal (Fig. 4a). Within the interstitium mononuclear leukocyteswere often seen in intimate contact with these fibroblast-likecells with ICAM-1 expression prominent at such sites of contact(Fig. 4b).

ICAM- 1 staining was also increased on proximal tubular cellscompared to normal, being localized exclusively to the brushborder (Fig. 5 a and b). ICAM-1 was not detected on the lateralor basal surfaces of these cells or within coated pits on the

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Fig. 3. ICAM-1 (CD54) in anti-GBM GN at 6hours. An "active-appearing" mononuclearleukocyte, probably a macrophage (M), isadherent to the endothelium of aperiglomerular interstitial capillary. ICAM-1 isstrongly expressed along the surface of thecapillary endothelium (asterisks) and ispresent at the site of leukocyte adhesion(arrow). ICAM-l is also more weakly andirregularly expressed on the surface of theleukocyte (arrowheads). Note ICAM-lstaining on the free surface of the glomerularparietal epithelium at the bottom of the figure.BC, Bowman's capsule. x 16,500.

brush border (Fig. 5 a and b). By 12 hours ICAM-l expressionwas also present on the luminal surface of distal convolutedtubules and collecting ducts. There was no evidence of leuko-cytic adhesion to tubular cells. Control sections demonstratedsome nonspecific gold staining solely in protein reabsorptiondroplets in proximal tubules.

In contrast to the relatively widespread expression ofICAM- 1, LFA-1 (CD! la and CD1 8) staining was observedsolely on the surface of mononuclear leukocytes. Both CD1 laand CD18 showed a similar pattern and intensity of expressionthroughout the tubulointerstitium. The "active-appearing"mononuclear leukocytes adherent to capillary endothelium(both macrophages and lymphocytes) showed strong surfaceexpression of LFA- 1, particularly at the sites of adhesion (Figs.6 and 7a). In contrast, nonadherent intracapillary leukocytesshowed little or no LFA-l expression (Fig. 7b). Cytoplasmicvacuoles within adherent macrophages contained positive stain-ing for LFA-1 (Fig. 6b). Mononuclear leukocytes were ob-served migrating through the capillary walls and extending longpseudopods within the interstitium. Such migrating leukocytesshowed strong LFA-1 expression along their entire surface(Fig. 8a). Mononuclear leukocytes in close contact with inter-stitial fibroblast-like cells also showed LFA-1 expression ontheir cytoplasmic membranes, particularly at the sites of adhe-

sion between the leukocytes and the fibroblast-like cells (Fig.8b). Control sections showed minimal nonspecific staining asdescribed for ICAM- 1.

Discussion

This is the first study to identify the ultrastructural localiza-tion of ICAM-l expression in glomerulonephritis. The promi-nent expression of ICAM-! and LFA-l antigens at sites ofadhesion between "active-appearing" leukocytes and intersti-tial capillary endothelium is consistent with the ICAM-1/LFA-linteraction playing a critical role in interstitial leukocytic infil-tration in experimental anti-GBM GN (Nikolic-Paterson et al,manuscript submitted). There was a clear up-regulation ofCD! la and CD18 antigens on the surface of these "active-appearing" leukocytes compared to nonadherent circulatingcells. The role of LFA-l appears to be in strengthening adhe-sion to activated endothelium following initial tethering involv-ing selectins [28, 29]. LFA-l expression on the surface oflymphocytes requires activation prior to mediating strong ad-hesion [29, 30]. Both ICAM-1 and LFA-l expression are alsoimportant in migration of mononuclear leukocytes through theendothelium [31—33], and this would be consistent with ourobservation that CD! la and CD18 are strongly expressed on the

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Fig. 4 (a) ICAM-1 (CD54) in anti-GBM GN at 6 hours. Amononuclear leukocyte (L) with sparse surface ICAM-l expression isadherent at several points (arrows) to an interstitial fibroblast-like cell(F). The fibroblast-like cell shows strong surface expression ofICAM-l including the sites of adhesion to the inflammatoryleukocyte. Note the absence of ICAM-l on the basal surface of theadjacent proximal convoluted tubule (PCT). x16,500. (b) ICAM-l(CD54) in anti-GBM GN at 12 hours. This lymphocyte (L) appears tobe tethered at several points to an interstitial fibroblast-like cell (F)which expresses strong surface ICAM-l. Note the positive ICAM-lstaining present at each of these sites of adhesion (arrows). x 18,000.

entire surface of leukocytes migrating both through the endo-thelium and within the interstitium. It is of interest that weobserved positive LFA- 1 staining within cytoplasmic vacuolesin macrophages which could represent part of a secretory orrecycling pathway for LFA- 1.

An important finding was that once the LFA-1 expressingleukocytes migrated into the renal interstitium, they localizedadjacent to, and demonstrated intimate contact with ICAM-lexpressing interstitial fibroblast-like cells. These fibroblast-likecells probably represent both interstitial fibroblasts and den-dritic cells which are difficult to distinguish solely on morpho-logic criteria [341. This leukocyte/fibroblast-like cell interactioncould have two possible functions. Firstly, these cell to cellcontacts could be an important mechanism by which mononu-

clear leukocytes develop focal localization within the renalinterstitium. Secondly, such cell interactions could allow pre-sentation of antigen to T lymphocytes. Such possibilities aresupported by studies demonstrating that ICAM-! expression onthe surface of cytokine stimulated dermal fibroblasts andLangerhan's cells in vitro is responsible for the binding ofactivated T lymphocytes [10, 35, 36], and that fibroblasts canexpress MHC-class II antigens in response to cytokine stimu-lation and are capable of acting as antigen presenting cells to Tlymphocytes [10, 37, 38]. In fact T lymphocyte proliferation inresponse to cells bearing low levels of MHC-class II moleculeexpression is greatly augmented by co-expression of ICAM-l[38].

Previous studies of this disease model found that interstitial

36 Hill et al: ICAM-1 in anti-GBM GN

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Hill et a!: ICAM-1 in anti-GBM GN 37

Fig. 5. ICAM-I (CD54) in anti-GBM GN at 24 hours. (a) ICAM-lexpression is confined to the brush border (asterisk) of the proximalconvoluted tubule (PCT). Note absence of staining on the lateral andbasal surfaces of the tubular cells (arrowheads). ICAM-l expressionis intense on the endothelial luminal surface of an adjacent interstitialcapillary (C, arrow). x 10,500. (b) At higher magnification ICAM-l isseen confined to the surface of microvilli of the brush border but isabsent from the coated pits (asterisks). x25,000.

leukocytic infiltration was evident in the periglomerular hilarregion at 12 hours after administration of anti-GBM serum.However, it was unclear whether these leukocytes had traf-ficked from the glomerulus down the mesangial stalk to this areaor if this represented a separate though related infiltrate [2].Using ultrastructural localization it is now evident that at leastpart of this initial hilar infiltrate derives from leukocytes migrat-ing out of periglomerular capillaries surrounding the hilar area.No leukocytic migration out of larger vessels was evident,suggesting that the initial "hilar arteriolar sheath" infiltrate [2]reflects solely an initial localization of leukocytes from thecapillaries in that area.

The rapid and strong up-regulation of ICAM-1 expression in

the periglomerular hilar region, the adhesion of "active-appear-ing" mononuclear leukocytes to nearby capillary endothelium,and leukocytic accumulation in the hilar region supports thepostulate that in this disease cytokines are produced within theglomerulus very rapidly following antibody deposition andpolymorphonuclear cell infiltration, and that such cytokines(IL-i and TNF, for example) may diffuse down the mesangialstalk or via the efferent glomerular circulation to the hilar areaand induce leukocytic infiltration [1, 3, 4, 39]. This postulate isfurther supported by the suppression of ICAM- 1 expression andinterstitial leukocyte infiltration by IL-i receptor antagonisttreatment in this disease model [40, Nikolic-Paterson et al,manuscript submitted]. We have demonstrated by immunogold

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Fig. 6. LFA-1 /3(CDJ8) in anli-GBM GN at 6 hours. (a) An adherent "active-appearing" mononuclear leukocyte, probably a macrophage (M),shows strong surface expression of CDI 8, particularly at the sites of adhesion to the endothelium of an interstitial capillary (arrows). Note absenceof any staining of endothelium or interstitial fibroblast-like cells (F). C, capillary lumen x 17,500. (b) At higher magnification CD18 is seen fairlyevenly distributed on the surface of this macrophage (M) adherent to the interstitial capillary endothelium (E). Note that CD18 is concentrated atthe points of leukocyte adhesion (arrows) and is also present within cytoplasmic vacuoles (asterisks). C, capillary lumen. X33,000.

electron microscopic localization that rabbit anti-rat GBMantibody in this model is localized exclusively to the glomerularbasement membrane and is not detectable within the tubuloin-terstitium in basement membranes surrounding Bowman's cap-sule, tubules or interstitial capillaries (Hill, unpublished obser-

vations). These findings, together with the absence of PMNL asa component of the interstitial leukocytic infiltrate suggest thatinterstitial heterologous antibody deposition and complementactivation do not play an important role in the interstitialleukocytic accumulation in this disease.

Hill et a!: ICAM-1 in anti-GBM GN 39

Fig. 7. LFA-1 a(CDIIa) in anti-GBM GN at12 hours. (a) Note the similar pattern andintensity of the staining of CD! la on thesurface of this leukocyte to that of CD18 inFig. 6a. This "active-appearing" mononuclearleukocyte, probably a macrophage, isadherent to the endothelium of an interstitialcapillary (C) in the periglomerular hilarregion. G, glomerulus; BC, Bowman'scapsule. x 10,500. (b) This nonadherentintracapillary mononuclear leukocyte,probably a lymphocyte (L), shows only verysparse surface CD! la as compared with thestrong pattern of staining on the adherentleukocyte in Fig. 7a. C, peritubular capillarylumen; PCT, proximal convoluted tubule.x 10,500.

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Fig. 8. LFA-J$ (CD 13) in anti-GBM GN at 6 hours. (a) Within the interstitium a migrating mononuclear leukocyte, probably a lymphocyte (L),expresses strong surface CD 18. The leukocyte is extending a long pseudopod (arrow) and its surface is in intimnte contact at several points withinterstitial fibroblast-like cells (F). C, interstitial capillary lumen; PCT, proximal convoluted tubule. x 14,000. (b) CD18 is strongly expressed onthe surface of this mononuclear leukocyte, prohably a lymphocyte (L), which shows a large area of contact with an interstitial fibroblast-like cell(F). Note CD18 is present on the leukocyte at the site of adhesion (arrows) but is not seen on the surface of the fibroblast-like cell. x20,000.

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The role of increased tubular ICAM-1 expression in theinitiation and progression of tubulointerstitial injury is not clear.Proximal tubular brush border expression of ICAM-l, which isonly very weak in normal kidneys, has been shown to beup-regulated in glomerulonephritis and graft rejection [17—21,25, 26, 41—43]. This was associated with increased tubularbasolateral MHC-class II antigen expression, and it has beenpostulated that the tubular cell may act as an antigen presentingcell promoting tubular immune damage 121, 25, 41—43]. In thisstudy we confirmed that ICAM- 1 is confined to the luminalmicrovillous surface of proximal tubules and is not detectableon the lateral or basal surfaces of these cells. In addition, by 12hours we demonstrated de nova expression of ICAM-1 on theluminal surface of distal tubules and collecting ducts. The solelyluminal localization of tubular ICAM-l expression raises thepossibility that it may be due to nonspecific reabsorption ofICAM-l filtered through a leaky glomerular barrier. The lack ofevidence of leukocytic adhesion to tubular cells suggests thatduring this initial phase of disease, up-regulation of tubularICAM-! expression plays no major role in interstitial leukocyticinfiltration or localization.

We conclude that ICAM-1 and LFA-1 play a role in the earlydevelopment of the tubulointerstitial inflammatory infiltrate inexperimental anti-GBM GN in the rat by (I) promoting theadhesion of mononuclear leukocytes within the interstitialcapillary bed, particularly in the periglomerular/perihilar re-gion, and their subsequent migration into the interstitium, and(2) by promoting interstitial localization of mononuclear leuko-cytes and their interaction with potentially antigen-presentinginterstitial fibroblast-like cells. The solely luminal localizationof tubular ICAM-l expression throws into doubt the importanceof tubular ICAM-1 in the development and progression oftubulointerstitial inflammation in the early stages of GN.

Acknowledgments

Dr. Hill was supported by a University of Melbourne ResearchFellowship. Part of this study was presented at the XIIth InternationalCongress of Nephrology in Jerusalem, June 1993. The authors thankMr. flarren Kelly and Miss Trudy Sawyer for technical assistance andMrs. Michelle Gough for preparation of the electron micrographs.

Reprint request to Dr. Prudence A. Hill, Department of Anatomy,The University of Melbourne, Parkville, Victoria 3052, Australia.

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