The Peri-islet Basement Membrane, a Barrier to Infiltrating Leukocytes in Type 1 Diabetes in Mouse and Human.

5.1 MB·English

by  Korpos, Eva

ORIGINALARTICLE
The Peri-islet Basement Membrane, a Barrier to
fi
In ltrating Leukocytes in Type 1 Diabetes in Mouse
and Human
Éva Korpos,1 Nadir Kadri,2,3 Reinhild Kappelhoff,4 Jeannine Wegner,1 Christopher M. Overall,4,5
Ekkehard Weber,6 Dan Holmberg,7 Susanna Cardell,2 and Lydia Sorokin1
We provide the first comprehensive analysis of the extracellular (3). However, once autoaggressive T cells are activated,
matrix (ECM) composition of peri-islet capsules, composed of severalstepsarecrucialfordiseaseprogression,including
theperi-isletbasementmembrane(BM)andsubjacentinterstitial extravasation of the CD4+, CD8+ T lymphocytes from the
matrix(IM),indevelopmentoftype1diabetesinNODmiceand postcapillaryvenulessurroundingtheisletsofLangerhans
in human type 1 diabetes. Our data demonstrate global loss of and their subsequent infiltration of the islets, leading to
peri-islet BM and IM components only at sites of leukocyte b-cell destruction and onset of disease symptoms. Pro-
infiltration into the islet. Stereological analyses reveal a correla- gression from the first steps in disease induction to the
tion between incidence of insulitis and the number of islets
presentation of symptomsoccursover a period of months
showing loss of peri-islet BM versus islets with intact BMs,
in NOD mice and several years in humans, with clinical
suggesting that leukocyte penetration of the peri-islet BM is
acriticalstep.Protease-andproteaseinhibitor–specificmicroar- symptoms appearing only when 70–90% of b-cells are
rayanalyses(CLIP-CHIP)oflaser-dissectedleukocyteinfiltrated destroyed, leaving a very narrow therapeutic window
andnoninfiltratedpancreaticisletsandconfirmatoryquantitative (4,5).
realtimePCRandproteinanalysesidentifiedcathepsinS,W,and In many cases, extravasated leukocytes can reside
Cactivityatsitesofleukocytepenetrationoftheperi-isletBMin around the islets of Langerhans without induction of dis-
association with a macrophage subpopulation in NOD mice and ease symptoms. Indeed, abundant infiltration has been
human type 1 diabetic samples and, hence, potentially a novel described in several experimental models that never or
therapeutic target specifically acting at the islet penetration
rarely progresses to disease (6,7), indicating that pene-
stage. Interestingly, the peri-islet BM and underlying IM are
tration of the pancreatic islet is a crucial step in disease
reconstituted once inflammation subsides, indicating that the
peri-islet BM-producing cells are not lost due to the inflamma- progression. Of all the steps in the progression of type 1
tion,whichhasimportantramificationstoislettransplantation diabetes, this final step from peri-islet infiltration to in-
studies. Diabetes 62:531–542, 2013 vasion of islets is the least well understood. However, to
understand the factors that convert a peri-islet infiltration
to an overt intra-islet infiltration that leads to diabetes
T would provide enormous potential for controlling disease
ype 1 diabetes is a chronic autoimmune disease progression and therapeutic development. The present
that targets the insulin-producing b-cells of the work addresses this important step in the development of
islets of Langerhans in the pancreas. Although it type1 diabetesinNOD miceandinhumans,inparticular,
is a T-cell (CD4 and CD8)–mediated disease, the contribution of the peri-islet basement membrane
macrophages,dendriticcells(DCs),andnaturalkillercells (BM) to this step.
are also essential for disease progression (1). NOD mice In all tissues, the extracellular matrix (ECM) acts to
are one of the best-studied animal models of type 1 di- separate tissue compartments but also provides specific
abetes (2) due to similarities with the pathogenesis and molecular signals that control processes such as cell mi-
genetics of human type 1 diabetes. The precise signal that gration,differentiation,andsurvival(8).Twomaintypesof
leads to disease initiation is not known and is considered ECM exist—the BM, which acts as a barrier limiting the
to be a combination of genetic and environmental factors movement of both soluble molecules and cells, and the
interstitial matrix (IM), which confers flexibility and elas-
ticitytotissues—bothofwhicharepresentintheperi-islet
From the 1Institute of Physiological Chemistry and Pathobiochemistry, Uni- capsule. Laminins and collagen type IV are the major
versityofMünster,Münster,Germany;the2DepartmentofMicrobiologyand
Immunobiology,UniversityofGothenburg,Gothenburg,Sweden;the 3De- constituents of the BM, each of which self-assemble to
partment of Microbiology, Tumor and Cell Biology, Karolinska Institute, form two protein networks that are interconnected by
Stockholm,Sweden;the4DepartmentofOralBiologicalandMedicalScien- nidogens and heparan sulfate proteoglycans, such as per-
ces,UniversityofBritishColumbia,Vancouver,BritishColumbia,Canada;
the5DepartmentofBiochemistryandMolecularBiology,CentreforBlood lecan (9). Collagen type IV is considered to be the struc-
Research, University of British Columbia, Vancouver, British Columbia, tural basis of BMs, whereas the laminins impart specific
Canada; the 6Institute of Biochemistry, Martin-Luther University of Halle- signalstocellsattachedtoormovingthroughBMsandare
Wittenberg,Halle,Germany;andthe7CenterforInfectionandInflammation
thereby responsible for biological activity. Laminins are
Research,CopenhagenUniversity,Copenhagen,Denmark.
Correspondingauthor:ÉvaKorpos,[email protected]. composed of an a, b, and g chain, and 5a, 4b, and 3g
Received10April2012andaccepted15August2012. laminin chains exist that combine to form at least 18 dif-
DOI:10.2337/db12-0432
ferent isoforms. These isoforms are differentially
This article contains Supplementary Data online at http://diabetes
.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0432/-/DC1. expressed in different BM types where they perform dif-
(cid:1)2013bytheAmericanDiabetesAssociation.Readersmayusethisarticleas ferent functions (10) mediated by direct binding to a vari-
longastheworkisproperlycited,theuseiseducationalandnotforprofit,
ety of different integrin and nonintegrin receptors (11).
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/fordetails. The IM layer, which in most tissues underlies the BM, is
diabetes.diabetesjournals.org DIABETES,VOL.62,FEBRUARY2013 531
PERI-ISLETMATRIXINTYPE1DIABETES
composedoffibrillarcollagens,suchascollagentypeIand Microscopy.Immunofluorescencestainingofcryosectionswasperformedas
III, nonfibrillar collagens, like collagen type VI, and non- described previously (26). For confocal microscopy of whole mounts, pan-
collagenous glycoproteins, like fibronectin, tenascins, creata were fixed in 4% paraformaldehyde and blocked in 1% BSA/0.3%
TritonX-100. The primary and secondary antibodies used are listed in
vitronectin, and chondroitin, or dermatan sulfate proteo-
SupplementaryTable1.SectionswereexaminedusingaZeissAxioImageror
glycans (12,13).
aZeissLSM510anddocumentedusingaHamamatsuORCAERcameraand
The IM and BM influence immune cell migration into or Volocity 5.4 software (Improvision). Samples for electron microscopy were
within inflamed tissues (14). The existing data, which are prepared according to standard protocols and analyzed with an EM-410
extremely limited, suggest that the biochemical nature of (Philips).
the ECM may determine the mechanism used by the im- Stereologicalanalysesandquantificationoftheperi-isletBMintegrity.
Frozen pancreata from 5-week-old (normoglycemic) and 14-week-old NOD
mune cells for its penetration, with protease-independent
miceweresectionedcompletelyandstainedwithantibodiestopan-lamininto
migration dominating (15–17). However, selective cleav-
labelBMs,CD45,apan-leukocytemarker,andinsulin.Atleast10healthyand
age mechanisms play a role in some cases (18,19). 10invadedpancreaticisletswereanalyzedpermousein5-and14-week-old
Limited information exists on the nature of the ECM of NOD mice (n = 3/group). Volocity 6.0.1 software was used to trace and
thepancreasand,inparticular,onthecompositionofperi- quantify (fluorescence intensity) the pan-laminin staining surrounding in-
islet capsule (20–22). Recent studies have reported the dividualpancreaticislets.Themeanfluorescenceintensitypermicrometerof
BM length was measured at sites showing continuous pan-laminin staining
existence of laminins, collagen type IV, and perlecan sur-
(SupplementaryFig.1AandC).Inpancreaticisletsshowingdisruptedpan-
rounding the pancreatic islets and the absence of staining lamininstainingatsitescoincidentwithCD45+cellsandlossofinsulin+cells,
for these antigens in pancreata of NOD mice, however, themeanfluorescencevaluepermicrometerwassignificantlyreduced(P,
withoutcorrelation todiseaseprogressioninmiceorwith 0.0007;SupplementaryFig.1BandC).Meanfluorescencevaluesofperi-islet
type 1 diabetes in humans (23,24). BMstainingforlamininwerethereforeusedtodefinehealthyversusinvaded
Here,weprovidethefirstcomprehensiveanalysisofthe pancreaticislets.
Forinvestigationofcorrelationsbetweenperi-isletBMintegrityanddisease
ECM of the peri-islet capsule, and the 3-dimensional re-
progression,frozenpancreatafrom5-,14-(normoglycemic)and21-week-old
lationshipamongtheperi-isletBM,peri-isletbloodvessels,
(diabetic)NODmiceweresectionedcompletely,andevery10thsectionwas
and peri-islet IM in NOD mice during the course of de- immunofluorescentlystainedwithpan-lamininantibodytolabelBMs,CD45,
velopmentoftype1diabetesandinsamplesfromhumans a pan-leukocyte marker, and insulin. Healthy islets with intact BMs were
recently diagnosed with type 1 diabetes. We demonstrate recordedwhennoinflammatorycellswerefoundaroundtheislet,peri-insulitis
global loss of peri-islet BM and IM components only at wasscoredwheninflammatorycellswerefoundaroundtheisletbuttheperi-
isletBMwasintact,andisletsshowingdisruptedpan-lamininstainingatsites
sites of leukocyte penetration of the peri-islet capsule,
coincidentwithCD45+cellsandlossofinsulin+cellswerescoredasinvaded
which contrasts to leukocyte extravasation from blood islets (300–400 islets were counted per mouse). Statistical analyses were
vesselsanddemonstratesfundamentaldifferencesinthese performedusingtwo-wayANOVA.
two processes. The number of islets showing loss of peri- LCM.Pancreatafrom 4-and 12-week-old NOD mice were snap frozen,12–
islet BM versus islets with intact BMs correlated with the 16 mm cryosections were cut and mounted on polyethylene naphthalate
incidence of diabetes, indicating that this is a crucial step membrane-coated slides (PALM MicroLaser Systems, Bernried, Germany),
in disease progression. Protease- and protease inhibitor– immediatelyfixed,dehydrated,andhematoxylinstainedusingtheHistoGene
Frozen Section Staining Kit (Arcturus). Inflamed and healthy islets were
specific microarray analyses (CLIP-CHIP) of laser-capture
excisedusingaLCMmicroscope(LeicaASLCM;Leica).
microdissection (LCM) material revealed upregulation of RNAextractionfromLCMsamples.TotalRNAwasisolatedusingthePico-
cathepsin S, W, and C in leukocyte-infiltrated islets com- PureRNAisolationKit(AppliedBiosystems,Carlsbad,CA),anditsintegrity
pared with noninvaded pancreatic islets. Parallel protein was assessed using the RNA 6000 Pico LabChipon in an Agilent 2100 Bio-
analyses revealed cathepsin S, W, and C expression in analyzer(AgilentTechnologies,SantaClara,CA).
AmplificationoftotalRNAandCLIP-CHIPmicroarrayanalysis.Owing
association with a subpopulation of macrophages at the
tothelowamountsofRNAisolated(7–16ng/mL),thecDNAlibrarygenerated
front ofleukocytepenetrationof theperi-islet BMin NOD fromRNAwasamplifiedusingtheTransPlexCompleteWholeTranscriptome
mice and in samples from humans with type 1 diabetes Amplificationkit(Sigma).ThecDNAwaspurifiedbytheGenElutePCRClean-
and, hence, potentially a novel therapeutic target specifi- Up Kit (Sigma) and applied to the CLIP-CHIP microarray, which was per-
callyactingattheisletpenetrationstage.Onceinflammation formedaspreviouslydescribed(27).
had subsided, the peri-islet BM and underlying IM were Quantitativereal-timePCR.Quantitativereal-timePCRwasperformedwith
35 ng amplified cDNA per sample in an ABI PRISM 7300 cycler (Applied
shown to be reconstituted in the mouse and human, in-
Biosystems,Darmstadt,Germany)usingBrilliantSYBRGreenQPCRMaster
dicating that the cells producing the peri-islet BM are not
Mix(AgilentTechnologies).Theresultswereinterpretedusingthecompara-
lost due to the inflammation, which has important ram- tiveCTmethod,wheretheamountofthetarget,normalizedtotheendoge-
ifications to islet transplantation studies. Our data suggest noushypoxanthine-guanine-phosphoribosyltransferasereferenceandrelative
that the ECM milieu influences the mode used by immune to a calibrator, is given by 22DDCT. Expression analysis specific for mouse
cells to infiltrate into tissues and raises novel possibilities cathepsin C, S, and W was performed. The unpaired t test was applied for
for tissue-specific immune modulatory therapies. statistics,andPvalues,0.05wereconsideredtobestatisticallysignificant.
In situ zymography. Matrix metalloproteinase (MMP)-2 and -9 activity in
pancreassectionswasdetected,aspreviouslydescribed(19).
RESEARCHDESIGNANDMETHODS
Animals. NOD mice were from Bomholtgaard (Ry, Denmark) and were RESULTS
screenedfordiabetesbyurineanalysesofglucosuria(Combur3Test;Roche).
ECMofthemurinepancreaticislet.Abroadrepertoire
Insulin pellets (LinShin Canada, Inc.) were inserted subcutaneously into di-
of antibodies specific for BM and IM proteins (Supple-
abeticNODmiceforlong-termstudies.Animalexperimentswereconducted
accordingtotheSwedishandGermananimalwelfareguidelines. mentary Table 1) were used to perform whole-mount im-
Humansamples.Humanhealthypancreasandtype1diabeticsampleswere munofluorescence staining of the pancreas and confocal
providedbytheNetworkforPancreaticOrganDonorswithDiabetes(nPOD) microscopy to generate a 3-dimensional view of the ECM
organbank. associatedwiththehealthypancreaticislet(Fig.1AandB;
Generation of anti-human laminin monoclonal antibodies. Mouse
Supplementary Video 1). Optical sectioning throughout
monoclonalantibodiesweregeneratedtohumanlaminina5,a4,a2,b1,b2,
entire islets, defined by insulin+ staining, revealed a con-
andg1chains.Antibodieswerecharacterizedbyenzyme-linkedimmunosor-
bent assay, tissue distribution as determined by immunofluorescence mi- tinuous BM encasing each islet that contained all major
croscopy,andbyWesternblotanalyses(25). BM components (collagen IV, perlecan, nidogens, and
532 DIABETES,VOL.62,FEBRUARY2013 diabetes.diabetesjournals.org
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FIG.1.A3-dimensionalprojectionofawholemountofahealthymousepancreasandvisualizationoftheperi-isletcapsulebyimmunofluorescence
stainingandelectronmicroscopyareshown.A:Insulin+andglucagon+pancreaticisletsareencasedintheperi-isletBM.B:Anopticalsectionof
a3-dimensionalprojectionofawhole-mountpancreaticisletshowsinasimultaneousviewofXY,XZ,andYZplanes,illustratingtheperi-isletBM
andendothelialBMs.Differentiallocalizationoflaminin(LM)a4anda5chainsinthepancreaticisletisshownbyimmunofluorescencestainingof
thinsections(C)andwhole-mountstaining(D).C:Meca32stainingmarkstheendotheliallayerofbloodvessels(insetsshowimagesathigher
magnification).E:CollagentypeIIIstainingvisualizestheIMoftheperi-isletcapsulethatunderliestheperi-capsularBM,markedbypan-laminin
(PLM)staining.F:ElectronmicrographyillustratestwoBMs:theperi-isletBM(arrow)thatenvelopstheb-cellsandtheendothelialBMofblood
vessels(arrowhead;inset1).Subjacenttoperi-isletBM(arrow),fibrillarcollagensareevident(*;inset2).Scalebarsare100mmforA–E,50mm
forinsetsinC,and1mminF.(Ahigh-qualitydigitalrepresentationofthisfigureisavailableintheonlineissue.)
diabetes.diabetesjournals.org DIABETES,VOL.62,FEBRUARY2013 533
PERI-ISLETMATRIXINTYPE1DIABETES
laminins; Table 1) (23). Examples of this peri-islet BM (Fig. 2B). However, the endothelial BM of blood vessels
staining are shown in Fig. 1A–D. A layer of IM occurred from the islet periphery and the acinar BMs remained in-
immediately subjacent to the peri-islet BM and was com- tact (Fig. 2A and B).
posed of the fibrillar collagens (types I and III; Fig. 1E), To quantify differences between the peri-islet BM of
together with several other typical interstitial matrix mol- healthy versus invaded islets, image analysis was used
ecules (Table 1). Electron microscopy confirmed that the to measure the fluorescence intensity of peri-islet pan-
pancreatic islet is ensheathed by a BM and an underlying laminin staining at sites were CD45 staining did and did
IM composed of fibrillar collagens (Fig. 1F), which to- not occur, revealing a statistically significant reduction in
gether comprise the peri-islet capsule. Electron micros- fluorescence intensity at sites of leukocyte invasion and
copy also revealed that endocrine cells within the loss of insulin+ cells within the islets (Fig. 2C; Supple-
pancreatic islet are associated with one BM, that of intra- mentaryFig.1).Toinvestigatewhetheracorrelationexists
isletbloodvessels,aspreviouslyreported(22,28),whereas between leukocyte penetration of the peri-islet BM and
theendocrinecellsattheisletborder,whichinthemouse progression of diabetes, a time-course study was un-
tend to be the glucagon+ a-cells (Fig. 1A), are associated dertaken. The number of islets showing disruption of the
with the peri-islet BM (Fig. 1F). BMwascomparedwiththenumberofisletswithanintact
Analyses of the laminin family of BM proteins together BM, as defined by fluorescence intensity of pan-laminin
with endothelial cell markers revealed that the peri-islet staining, in NOD mice aged 5 (presymptomatic stages), 14
BM was biochemically distinct to the BMs surrounding (early onset), and .21 weeks (diabetic stage). Stereolog-
bloodvesselsandcontainedlaminina2,a4,b1,b2,g1,g2, ical analyses of these immunofluorescence data revealed
and g3 chains (Fig. 1C and D) but not laminin a1, a3, and that the number of islets showing reduced insulin staining
a5 chains (Fig. 1D). By contrast, blood vessel BMs within and presence of invading CD45+ cells and, therefore, de-
andsurroundingisletscontainedlaminina4anda5chains fined as intrainsulitis, increased with age and revealed an
(Fig. 1C and D) together with laminin g1 and b1 chains. inverse correlation between disease progression and the
Laminin a5 expression was stronger in the arterioles integrityoftheperi-isletBM(Fig.2BandD).In5-week-old
where smooth muscle cells express this chain in addition NOD pancreata, most islets were intact, few showed peri-
to endothelial cells (Fig. 1D). The closeness of blood ves- insulitis, and islets with disrupted BMs were not present.
selstotheperi-isletBM,surroundingtheisletsorentering This ratio changed in 14-week-old NOD pancreata, and
them,resultedintheappearanceofareasmoreintenseBM ,50%oftheisletswereinvadedbyleukocytesandshowed
staining (Fig. 1A, B, and D). Parallel analyses of the ex- degraded BMs. In 21-week-old NOD pancreata, 80% of the
pressionofthemajorECMreceptorsrevealedlocalization islets showed loss of peri-islet BM and IM staining (Fig.
of Lutheran blood group glycoprotein and b-dystroglycan 2D). Taken together, these data suggest a correlation be-
on endothelial cells and a weak immunosignal for b- tween degradation of the peri-islet capsule and disease
dystroglycan on cells that were close to the peri-islet BM progression and that penetration of the peri-islet BM is
(Table1).AsummaryoftheBMandIMcomponentsofthe a critical step in disease progression.
peri-islet capsule and their receptors are listed in Table 1. Mechanism of loss of the peri-islet BM and IM in the
Loss of the peri-islet capsule in the inflamed NOD NODpancreas.Owingtothedifferentbiochemicalnature
pancreas. Optical sectioning of immunofluorescently of BM and IM proteins, the complete loss of the peri-
stained whole-mount inflamed NOD pancreata suggested capsular ECM at sites of leukocyte penetration into the
a striking loss of all BM and IM components of the peri- islet suggests the involvement of several different pro-
islet capsule specifically at sites of leukocyte infiltration teases or of proteases with broad substrate specificity. To
into the pancreatic islets (Fig. 2A and B; Table 1; Supple- investigate whether proteases were involved, analyses
mentary Video 2) and loss of insulin staining within islets were performed with the murine CLIP-CHIP microarray
TABLE1
SummaryofECMcomponentsofthehealthyandleukocyte-infiltrated peri-isletcapsuleinNODmice
Pancreaticislet Pancreaticislet
BMcomponents Healthy Infiltrated IM components Healthy Infiltrated
Laminina1 – – Fibronectin + –
Laminina2 + – CollagentypeI + –
Laminina4 + – CollagentypeIII + –
Laminina5 – – CollagentypeVI + –
Lamininb1 + – Fibrillin-1 – –
Lamininb2 + – Fibrillin-2 + –
Lamining1 + – Matrilin-2 + –
Laminin332* + – ERTR7antigen + –
CollagentypeIV + – Tenascin-C – –
Perlecan + – ECMreceptors
Agrin + – b-Dystroglycan +/2 –
Nidogen-1 + – Lutheranblood group – –
glycoprotein
Nidogen-2 + – Integrinb1 + –
Integrinb4 +/2 –
–,notpresent;+,present;+/2,weak.*Antibodydoesnotdistinguishbetweenthethreechainsconstitutingthisisoform.
534 DIABETES,VOL.62,FEBRUARY2013 diabetes.diabetesjournals.org
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FIG.2.Penetrationoftheperi-isletBMisacriticalstepindiseaseprogressioninNODmice.Immunofluorescencestainingofawholemount(A)
andthinsections(B;boxedareasshowhighermagnification)ofleukocyte-invadedisletsforpan-laminin(PLM)asamarkeroftheperi-isletBM,
andthepan-leukocytemarkerCD45illustratesthecorrelationbetweenthelossoftheperi-isletBMandleukocyteinfiltrationintothepancreatic
islet.Theperi-isletBMremainsdetectable(arrows)atsitesofleukocyteaccumulationaroundtheisletbutnotwhereleukocyteshaveinvadedthe
islet(arrowhead).C:Quantificationofperi-isletBMintegritywasperformedbymeasuringthemeanfluorescenceintensitypermicrometerofthe
peri-isletBM(10islets/mouse,n=3/eachgroup).Themeanfluorescenceintensitypermicrometerofdegradedperi-isletBMsissignificantlylower
thanthatofintactperi-isletBMs.***P<0.0007.D:Stereologicalanalysesofpancreaticsectionsfrom5-and14-week-oldnormoglycemicand21-
week-olddiabeticNODmice(n=3/eachgroup)wereusedtoquantifythenumberofisletswithintactperi-isletBMs(healthyisletsandisletsin
peri-insulitisstage)vs.invadedisletsshowinglossofperi-isletBMstaining.W,weeks.Scalebarsare100mmand50mmininsets(P<0.0001).(A
high-qualitydigitalrepresentationofthisfigureisavailableintheonlineissue.)
diabetes.diabetesjournals.org DIABETES,VOL.62,FEBRUARY2013 535
PERI-ISLETMATRIXINTYPE1DIABETES
(29). LCM was used to isolate healthy islets from 4-week shown here to correlate with sites of loss of peri-islet BM
NODpancreata,andisletsshowingearlystagesofimmune (Fig. 4A). Immunofluorescence staining revealed some
cell penetration were isolated from .12-week NOD pan- colocalization of F4/80+ macrophages with cathepsin C
creata; mRNA was purified, and gene expression patterns staining (Fig. 4B) and more extensive colocalization with
were compared (Table 2; Supplementary Fig. 2). Data re- cathepsin S and W (Fig. 4C and D), suggesting that
veal upregulation of caspase 7 and 8 in infiltrated islets, a macrophage subpopulation may be one of the cellular
which are members of the aspartic-specific cysteine pro- sources that secrete these proteases at the site of leuko-
tease family associated with apoptotic events (30). This is cyte penetration of the peri-islet BM. A restricted colo-
in accordance with enhanced b-cell apoptosis due to au- calization of cathepsin S and W with some CD11c+ DCs
toimmune attack, thereby validating our sample prepara- was observed (data not shown).
tion. In addition, several members of the MMP family, Peri-islet BM degradation in type 1 diabetic human
including MMP-2, MMP-7, and MMP-14, were upregulated pancreas. To investigate whether leukocyte infiltration
atthemRNAlevelininfiltratedislets.However,thiscould into the human pancreatic islets is also associated with
notbeconfirmedattheproteinlevelbecauseinsitugelatin loss of peri-islet ECM components, human type 1 diabetic
zymography for the detection of MMP-2, MMP-9, and par- samples were analyzed (Supplementary Tables 2 and 3).
tiallyalsoforMMP-14(becauseitisanupstreamactivator As in NOD samples, the healthy human pancreatic islet
of MMP-2) (31) revealed gelatinase activity in association was surrounded by a capsule composed of a peri-islet BM
withtheinsulin-producingb-cellsinthehealthypartofthe and subjacent IM (Fig. 5A–C). Although most ECM pro-
isletratherthanatsitesofleukocyteentry(Supplementary teins analyzed showed the same localization pattern as
Fig.3).ElevatedMMP-7activity,whichcanbedetectedby observedinNODpancreata,onlylaminina5wasfoundto
casein gel zymography, could also not be confirmed in mark the peri-islet BM, which was not the case in mouse
islets showing leukocyte infiltration (data not shown). samples, a result consistent with findings from previous
Several members of the cathepsin protease family, in- studies (21,22). Sections from the pancreas head, body,
cludingcathepsinC,H,S,andW,showedafour-tosixfold and tail were analyzed by immunofluorescence staining.
increaseinmRNAexpressionininfiltratedislets(Table2), DatashowninFig.5D–Harefromonesample(nPODcase
which was confirmed by quantitative real-time PCR (Sup- 6052),a12-year-olddonorwithtype1diabeteswhodied1
plementary Fig. 4). This was unusual, because cathepsins week after diagnosis, which still contained some insulin+
are best known as intracellular proteases; however, there islets surrounded by leukocytes, according to the nPOD
is evidence that they can also act extracellularly (32). histological analysis (http://www.jdrfnpod.org/). Insulin+
Consistent with this possibility was the upregulation of isletsoccurredonlyinthepancreaticbodyandtailwhere,
several members of a-1 antitrypsin family (Table 2), as in NOD samples, sites of leukocyte infiltration of the
known to inhibit cysteine proteases as well as serine pancreatic islet were associated with loss of peri-islet BM
proteases (33). We therefore used immunofluorescence staining(Fig.5EandF;SupplementaryTable3);however,
microscopy to investigate whether cathepsins C, S, and W no changes were observed in the case of peri-insulitis
2
colocalized with the islet-invading leukocytes. In situ lo- alone. In the pancreatic head, only insulin islets oc-
calization of all three cathepsins correlated with sites of curred, which were associated with little or no CD45+
loss of peri-islet BM staining in NOD mice (Fig. 3A–C). cells, indicating that they represented islet remnants. As
MacrophagesandDCsareamongthefirstcellsrecruited fortheNODmouse,triple-immunofluorescencestainingin
to pancreatic islets (34,35), and their localization was samples from humans with type 1 diabetes revealed that
TABLE2
SummaryofmurineCLIP-CHIPmicroarrayanalyses
MEROPS identifier Gene Description Referencesequences Foldchange*
C14.004 Casp7 Caspase7 NM_007611 3.34
C14.009 Casp8 Caspase8 NM_009812 3.55
C01.070 Ctsc CathepsinC NM_009982 4.57
C01.040 Ctsh CathepsinH NM_007801 4.33
C01.034 Ctss CathepsinS NM_021281 6.32
C01.037 Ctsw CathepsinW NM_009985 4.84
M10.003 Mmp2 GelatinaseA NM_008610 5.33
M10.008 Mmp7 Matrilysin NM_010810 2.90
M10.014 Mmp14 Membranetype NM_008608 3.73
1-MMP
I04.xxx Serpina1b a1-antitrypsin NM_009244 10.56
member1b
I04.xxx Serpina3g a1-antitrypsin XM_484175 16.12
member3g
I04.xxx Serpina1a a1-antitrypsin NM_009243 24.42
member1a
I04.xxx Serpina1d a1-antitrypsin NM_009246 17.85
member1d
I35.002 Timp2 Tissueinhibitorof NM_011594 3.67
metalloproteases-2
*FoldchangesinproteaseandproteaseinhibitormRNAlevelsinisletsshowingearlyinfiltrationcomparedwithisletsshowingnoleukocyte
infiltrationinNODmice.
536 DIABETES,VOL.62,FEBRUARY2013 diabetes.diabetesjournals.org
É.KORPOSANDASSOCIATES
FIG.3.ValidationofCLIP-CHIPdataattheproteinlevelbyimmunofluorescenceinNODsamples.ImmunofluorescencestainingofNOD21-week-
oldpancreassectionsforcathepsinC(A),cathepsinS(B),andcathepsinW(C)showslocalizationatsiteswhereperi-isletBMstainingislost
(arrows),asshownherebylaminin(LM)g1staining.Theboxedareasareshownathighermagnificationtotheright.Scalebarsare100mm.(A
high-qualitydigitalrepresentationofthisfigureisavailableintheonlineissue.)
someoftheCD45+cellswerealsopositiveforcathepsinS proteins. In both human and NOD samples, glucagon+
andWstainingatsitesofperi-isletBMloss(Fig.5IandJ). a-cell clusters were closely associated with the peri-islet
Reconstitution of the peri-islet BM in long-term BM,whichwerelocatedmainlyalongtheperipheryofthe
diabetic NOD mice and type 1 diabetic patients. Sur- islet (Fig. 5H, Fig. 6; Supplementary Video 3). During the
prisingly, in several human diabetic pancreas samples we course of inflammation in NOD mice, a-cells formed
observedmanyisletsthatwereinsulin2/glucagon+butalso clusters that were encased by a BM of the same compo-
lacked CD45+ leukocytes and were encased in an intact sitionastheperi-isletBMandalsosurroundedbyIM(Fig.
peri-islet BM (Fig. 5G and H). Such islets dominated the 6A–C). This suggests that despite the massive infiltration
pancreata of donors who had been diagnosed with the of leukocytes, a-cellinteractionwiththe BMcan protectit
disease for several weeks (nPOD cases 6062–6064; Sup- fromdegradation.Whetherthisisduetodirectsecretionof
plementaryTable2)andtype1diabeticmedalistssamples ECM components by the a-cells or to their cross talk with
that were diagnosed with diabetes for more than 50 years other peri-islet cells that secrete ECM is not yet clear.
(nPOD cases 6085 and 6086). These results suggest that Nevertheless,thedatasuggestthatcellularinteractionwith
the cells responsible for producing the peri-islet BM are the peri-islet BM is required for its integrity and barrier
not lost due to the inflammation and can reconstitute the function.
peri-islet BM once the inflammation has subsided. We
show here that this reconstituted peri-islet BM in human
samples of type 1 diabetes contains laminin a5 (Fig. 5H), DISCUSSION
which has been shown to be a ligand for b-cells, in- Weshowthattheperi-capsularECMiscomposedofaperi-
teraction with which enhances insulin production (28). islet BM embedded in an IM layer that interconnects with
Hence, reconstitution of the peri-islet BM may have im- the surrounding blood vessels and exocrine tissue. The
portant ramifications to islet transplantation studies. We composition of the peri-islet BM is biochemically distinct
therefore used diabetic NOD mice (aged .21 weeks) to BMs of blood vessels surrounding or within the islet,
supplemented with subcutaneous insulin pellets to in- with characteristic expression of laminin a4 in the mouse
vestigate whether reconstitution of the peri-islet BM in- and of laminin a5 in human tissue (21,22). Quantitative
deed occurs after inflammation has subsided and what is analysesrevealedsignificantlyreducedmeanfluorescence
the potential cellular source or sources of these BM intensityoftheperi-isletBMpan-lamininstainingatsitesof
diabetes.diabetesjournals.org DIABETES,VOL.62,FEBRUARY2013 537
PERI-ISLETMATRIXINTYPE1DIABETES
FIG.4.Macrophagesareapotentialcellularsourceofcathepsinsinleukocyte-invadedpancreaticisletsinNODmice.A:Immunofluorescence
stainingrevealsF4/80+macrophagesclosetothesiteswhereperi-isletBMstainingislost(arrows),asshownherebypan-laminin(PLM)staining.
Theboxedareaisshownathighermagnificationtotheright.Triple-stainingforF4/80,asamarkerformacrophages,thenucleimarkerDAPIand
cathepsinC(B),cathepsinS(C),orcathepsinW(D)showcolocalizationofallthreecathepsins,withasubpopulationofmacrophages.Scalebars
are100mm.(Ahigh-qualitydigitalrepresentationofthisfigureisavailableintheonlineissue.)
leukocyteinvasionofisletsandlossofinsulin+cellsandthat of upregulation of several members of the cathepsin pro-
this correlates with disease progression, with a higher in- teasefamilyandproteaseinhibitorsinleukocyte-infiltrated
cidenceofinsulitiswhenmoreisletsshowedpenetrationof islets. In vivo staining for cathepsin S, C, and W showed
the peri-islet BM. This indicates that leukocyte penetration colocalizationwithsomemacrophagesattheinflammatory
of the peri-islet BM is a critical step in disease progression front, suggesting their potential secretion from a macro-
and potentially represents a new therapeutic target. phagesubpopulationbutnotexcludingothersources.This
The widespread disappearance of IM and BM compo- is consistent with the exclusive expression of cathepsins
nents from the mouse and human islet capsule indicates S and W by inflammatory cells (36,37) and broad cellular
that leukocyte infiltration into the pancreatic islet is fun- expressionofcathepsinC(38),andmayexplainwhyonly
damentallydifferentfromthemainlynonproteolyticmode the BM and IM components of the peri-islet capsule are
ofleukocyteextravasationfrompostcapillaryvenules(14). selectively lost and not BMs of peri-islet blood vessels or
Italsosuggests theinvolvement of severalproteases orof the acini of the exocrine pancreas, despite widespread
proteases with broad specificity. CLIP-CHIP microarray an- leukocyte infiltration. Further, cathepsin S–null mice on a
alyses support the latter possibility, with the identification NOD background show significantly reduced incidence of
538 DIABETES,VOL.62,FEBRUARY2013 diabetes.diabetesjournals.org
É.KORPOSANDASSOCIATES
FIG.5.ECMofhumanhealthyandtype1diabeticpancreata.A:ImmunofluorescencestainingrevealstheBMcomponentlaminin(LM)a5sur-
roundingtheinsulinandglucagon-containingislet.Glucagon+cellsareorganizedalongtheperi-isletBM,whereastheinsulin+b-cellsfilltheinner
partoftheislet.B:Double-stainingforpan-laminin(PLM)andotherBMcomponents,suchasperlecan,showsomecolocalizationintheperi-islet
BM(arrow)andintheendothelialBM(arrowhead).C:Theperi-isletBMisfurthersurroundedbyadensenetworkofIM,representedhereby
collagentypeIII.(A–C,nPODcase6024,healthypancreas)D:Schematicrepresentationoftheareasanalyzed.Inarecentlydiagnosedindividual
withtype1diabetes(nPODcase6052),insulin+/CD45+isletsweredetectedonlyinthepancreatictail(EandF)butnotinthehead(G)ofthe
pancreas,whereonlyinsulin2(G)andglucagon+(H)isletswithintactBMswerepresent.Theperi-isletBMisintactatsiteswheretheleukocytes
accumulatearoundtheinsulin+islets(arrowsinE)andislostatsitesofleukocyteinvasion(arrowheadsinEandF).H:Glucagon-producing
a-cellsbecomeevenlydistributedthroughouttheisletintype1diabeticCD452/insulin2samplesandareenclosedwithinanintactperi-isletBM,as
shownbylaminina5staining.IandJ:Stainingofsamplesfromindividualswithrecentlydiagnosedhumantype1diabetesshowlossoftheperi-
isletBMatthefrontoftheCD45+cellinfiltration(arrows)andcolocalizationofimmunoreactivityforcathepsinS(I)andforcathepsinW(J).The
peri-isletBMismarkedherebyperlecanstaining.Theboxedareasareshownathighermagnificationstotheright.Scalebarsare100mm.(Ahigh-
qualitydigitalrepresentationofthisfigureisavailableintheonlineissue.)
diabetes.diabetesjournals.org DIABETES,VOL.62,FEBRUARY2013 539
PERI-ISLETMATRIXINTYPE1DIABETES
diabetes (39). This has been attributed to cathepsin S ef- laminins a4 or a5 is essential for growth and strongly
fectsonantigenpresentation(40),butaroleinpenetration promotesinsulinsecretionviaintegrin-mediatedprocesses
of the peri-islet BM cannot be excluded. (28). Taken together this suggests that inclusion of such
AlthoughMMPs,inparticularMMP-2andMMP-14,were peri-islet BM-specific laminins or the integrin-binding
also upregulated at the transcriptional level, in situ domains thereof, or cells capable of regenerating the BM,
zymography demonstrated the absence of correlation be- wouldenhanceislettransplantationsuccess.Althoughour
tweengelatinaseactivityandsitesofleukocyteinfiltration. data highlightthe a-cellsas apotential source of peri-islet
Rather, gelatinase activity was associated with healthy, BMcomponentsbecauseoftheirtightassociationwiththe
insulin-producing portions of the islets. The upregulation peri-islet BM in the reconstituted islets, other peri-islet
of MMP-14 is consistent with its proposed role in T-cell cells, such as myofibroblasts or Schwann cells, which can
extravasationintothepancreasviaCD44cleavage(41)but synthesizeBMandIMcomponents,mayalsocontributeto
canbeexcludedasacandidatefordegradationoftheperi- peri-islet BM restoration and represent appropriate sup-
islet capsule. The upregulation of several protease inhib- port cells for islet transplantation.
itors,includingtissueinhibitorofmetalloproteinases-2and In conclusion, our data demonstrate that leukocyte pen-
the serpins (a1-antitrypsin members), broad specificity etration oftheperi-isletBMisacriticalstepinprogression
proteinase inhibitors, suggests a compensatory mecha- of type 1 diabetes, the mechanism of which differs funda-
nismtocounteractthedestructiveeffectofproteases(33), mentallyfromthatinvolvedinleukocyteextravasationfrom
the balance of which probably controls whether the peri- bloodvessels.ThissuggeststhattheECMmilieuinfluences
isletBMcanbepenetrated.Thestrongupregulationofthe the mode used by immune cells to infiltrate into tissues
serpins shown here is also consistent with the reported and raises novel possibilities for tissue-specific immune-
anti-inflammatory effects in NOD mice (42) and its recent modulatorytherapies.Weidentifycathepsinsatthefrontof
useinclinicaltrialsfortreatmentoftype1diabetes(http:// leukocytepenetrationoftheperi-isletBMinNODmiceand
clinicaltrials.gov/ct2/show/NCT01319331). intype1 diabetic samples from humans and, hence, poten-
Precisely how the cathepsins function in leukocyte pene- tially a novel therapeutic target specifically acting at the
tration of the peri-islet BM is not clear. The cathepsins are islet-penetrationstage.Demonstrationthattheperi-isletBM
knownfortheirterminalproteolyticactivityinlysosomesbut andunderlyingIMcanbereconstituted,onceinflammation
have been shown to be secreted in certain circumstances hassubsidedinthemouseandhuman,opensnewavenues
and can be active at neutral pH in an extracellular milieu for the use of peri-islet BM components or BM-producing
(43). Human macrophages can also create a pericellular cells in islet transplantation strategies.
acidic microenvironment by producing vacuolar-type H+-
ATPase components, thus creating an optimal micromilieu
for cathepsin activity solely in their close vicinity (44). ACKNOWLEDGMENTS
Cathepsins have also been reported to cleave ECM mole- ThestudyreceivedsupportfromtheEuropeanFoundation
cules;inparticular,cathepsinShasbeenshowntocleave for the Study of Diabetes/Juvenile Diabetes Research
Matrigel, a mixture of BM components (45), as well as Foundation (JDRF)/Novo Nordisk A/S (BD21070), JDRF
laminins g2 and b1 (46) and collagen type IV (47), all of (1-2005-903).Thisresearchwasperformedwiththesupport
whicharepresentintheperi-isletBM.Further,cathepsin of nPOD, a collaborative type 1 diabetes research project
S has been reported to cleave the native triple-helical sponsored by the JDRF. Organ Procurement Organiza-
regionoffibrillarcollagentype I(48),whichispresentin tionspartneringwithnPODtoprovideresearchresources
the peri-islet IM. Hence, the possibility exists for a direct are listed at http://www.jdrfnpod.org/our-partners.php.
involvement of cathepsins in degradation of the peri-islet No other potential conflicts of interest relevant to this
ECM. However, an indirect involvement of cathepsins by article were reported.
initiating a proteolytic cascade resulting in activation of É.K.performedallexperimentsandwasinvolvedinwrit-
otherproteases that degradetheECMis also possible. In ing the manuscript. N.K. provided advice on immunology,
addition, cathepsins may be associated with degradation contributed to discussion, and reviewed the manuscript.
of endogenous inhibitors, resulting in increased activity R.K. and C.M.O. helped in the microarray analyses, with
of other proteases (49). the CLIP-CHIP analysis, and with statistical analyses of
Interestingly,insamplesfromhumanswithlong-termtype the data. J.W. performed the quantitative real-time PCR
2
1diabetes,wefoundinsulin isletssurroundedbyanintact experiments. E.W. provided antibodies and advice on the
peri-isletBM and underlying IM. By implanting insulin cap- cathepsins.D.H.andS.C.wereinvolvedinprojectdevelop-
sules intodiabeticNOD mice, wewereabletoobservethe ment and contributed significantly to compilation of the
same phenomena and also identify the early formation of manuscript.L.S.supervisedtheworkdoneinthelaboratory
a-cell,glucagon+clustersthatweresurroundedbyaBMof and participated inwriting the manuscript. L.S. isthe guar-
thesamecompositionastheperi-isletBM.Thissuggeststhat antor of this work, and, as such, had full access to all the
the cells responsible for formation of the peri-islet BM are data in the study and takes responsibility for the integrity
notlostduetotheinflammationandcanregeneratetheBM of the data and the accuracy of the data analysis.
once the inflammation has subsided. Given that trans- The authors thank Hartmut Halfter (Department of
plantation experiments have highlighted the importance of Neurology, University of Münster, Münster, Germany) for
theregenerativeabilityoftheperi-isletcapsuletolong-term use of the LCM; Friedemann Kiefer, Stefan Volkery, and
graft survival, this is a significant finding. The ECM sur- Ruben Böhmer (Max Planck Institute for Molecular Medi-
rounding the islet is destroyed during islet isolation for cine, Münster, Germany) for assistance in the use of the
transplantation experiments, leading to integrin-mediated LSM510 microscope; Karin Loser (Department of Derma-
cell death; however, preservation of an ECM mantel signif- tology,UniversityofMünster,Münster,Germany)forhelp
icantlyincreasesthesurvivalrateoftransplantedislets(50). with quantitative real-time PCR; Horst Robenek and Karin
More recently, developmental studies in the mouse Schlattmann (Arteriosclerosis Research, University of
have shown that b-cell adhesion to the BM components Münster, Münster, Germany) for help in the use of the
540 DIABETES,VOL.62,FEBRUARY2013 diabetes.diabetesjournals.org