Induction of resistance to diabetes in non-obese diabetic mice by targeting CD44 with a specific monoclonal antibody.

Abstract

Inflammatory destruction of insulin-producing beta cells in the pancreatic islets is the hallmark of insulin-dependent diabetes mellitus, a spontaneous autoimmune disease of non-obese diabetic mice resembling human juvenile (type I) diabetes. Histochemical analysis of diabetic pancreata revealed that mononuclear cells infiltrating the islets and causing autoimmune insulitis, as well as local islet cells, express the CD44 receptor; hyaluronic acid, the principal ligand of CD44, is detected in the islet periphery and islet endothelium. Injection of anti-CD44 mAb 1 hr before cell transfer of diabetogenic splenocytes and subsequently on alternate days for 4 weeks induced considerable resistance to diabetes in recipient mice, reflected by reduced insulitis. Contact sensitivity to oxazolone was not influenced by this treatment. A similar antidiabetic effect was observed even when the anti-CD44 mAb administration was initiated at the time of disease onset: i.e., 4-7 weeks after cell transfer. Administration of the enzyme hyaluronidase also induced appreciable resistance to insulin-dependent diabetes mellitus, suggesting that the CD44-hyaluronic acid interaction is involved in the development of the disease. These findings demonstrate that CD44-positive inflammatory cells may be a potential therapeutic target in insulin-dependent diabetes.

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Induction of resistance to diabetes in non-obese
diabetic mice by targeting CD44 with a specific
monoclonal antibody
Lola Weiss*, Shimon Slavin*, Shoshana Reich*, Patrizia Cohen†, Svetlana Shuster‡, Robert Stern‡, Ella Kaganovsky§,
Elimelech Okon§, Ariel M. Rubinstein¶, and David Naor¶?
*Department of Bone Marrow Transplantation and Cancer Research Laboratory, Hadassah University Hospital, Jerusalem 91120, Israel;†Department of
Pathology, Hadassah University Hospital, Mount Scopus, Jerusalem 91240, Israel;‡Department of Pathology, School of Medicine, University of California,
San Francisco, CA 94143-0506;§Department of Pathology, Rabin Medical Center, Petah Tikva, 49100 Israel;¶Lautenberg Center for General and Tumor
Immunology, Hebrew University-Hadassah Medical School, POB 12272, Jerusalem 91120, Israel
Edited by Anthony Cerami, The Kenneth S. Warren Laboratories, Tarrytown, NY, and approved November 15, 1999 (received for review August 19, 1999)
Inflammatory destruction of insulin-producing ? cells in the pan-
creaticisletsisthehallmarkofinsulin-dependentdiabetesmellitus,
a spontaneous autoimmune disease of non-obese diabetic mice
resembling human juvenile (type I) diabetes. Histochemical anal-
ysis of diabetic pancreata revealed that mononuclear cells infil-
trating the islets and causing autoimmune insulitis, as well as local
islet cells, express the CD44 receptor; hyaluronic acid, the principal
ligand of CD44, is detected in the islet periphery and islet endo-
thelium. Injection of anti-CD44 mAb 1 hr before cell transfer of
diabetogenic splenocytes and subsequently on alternate days for
4 weeks induced considerable resistance to diabetes in recipient
mice, reflected by reduced insulitis. Contact sensitivity to ox-
azolone was not influenced by this treatment. A similar antidia-
betic effect was observed even when the anti-CD44 mAb admin-
istration was initiated at the time of disease onset: i.e., 4–7 weeks
after cell transfer. Administration of the enzyme hyaluronidase
also induced appreciable resistance to insulin-dependent diabetes
mellitus, suggesting that the CD44–hyaluronic acid interaction is
involved in the development of the disease. These findings dem-
onstrate that CD44-positive inflammatory cells may be a potential
therapeutic target in insulin-dependent diabetes.
T
immunological response, release of chemokines, cytokines, and
toxic agents (e.g., reactive oxygen) by the activated cells, stim-
ulation of endothelial cells, up-regulation of cell surface adhe-
sion molecules, transendothelial cell migration, and a shift in the
Th1?Th2 balance in favor of the Th1 cells (1). Hence, the
destructiveautoimmuneinflammatoryprocessdependssubstan-
tially (but not exclusively) on cell migration and cell interaction
with matrix components of target organs. The destruction of
pancreatic islet ?-cells in insulin-dependent diabetes mellitus
(IDDM) by invading leukocytes and the consequent deteriora-
tion of the insulin-dependent glucose homeostasis is an excellent
example of such an autoimmune process (2, 3), although neither
the nature of the triggering self-antigen nor the molecules
associated with its recognition and presentation have been
unequivocally identified.
Whereas the function of selectins and integrins in supporting
inflammatory cell migration and lodgment has been well estab-
lished (4), the role of cell surface CD44 has only recently attracted
attention (5). Alternative splicing and?or posttranslation modifica-
tions generate many CD44 isoforms. The large array of CD44
isoforms is mainly attributable to the insertion of amino acid
sequences, encoded by different combinations of 10 variant exons,
into a membrane proximal position of the extracellular domain.
Transcripts in which these variant exons are spliced out encode the
most common and widely expressed 85-kDa isoform, known as
standard CD44. The expression of CD44 isoforms containing
sequences encoded by the variant exons (CD44v) is tightly regu-
he inflammatory cascade in affected organs of autoimmune
diseases is a complex process that involves triggering of the
lated. Expression of CD44v isoforms is restricted to epithelial cells
and cells undergoing activation or differentiation, as well as some
progressor tumors. CD44 isoforms are involved in a wide variety of
cellularprocesses,suchascell–cellandcell–matrixinteractions,cell
trafficking, presentation of cytokines and growth factors to travel-
ing cells, and the uptake and intracellular degradation of hyaulo-
ronicacid(HA),theprincipalligandofthisreceptor.Inadditionto
HA, other tissue and cell components (e.g., collagen, fibronectin,
laminin, serglycin, and osteopontin) can interact with CD44 (re-
viewed in refs. 5 and 6).
Although the triggering mechanism of IDDM remains elusive,
it is clear that the entire process depends on the migration of
inflammatory cells into the pancreatic islets and their interaction
withtheirmatrix.BecauseCD44isassociatedwithbothactivities
(5), we decided to study its involvement in experimental IDDM
in non-obese diabetic (NOD) mice by exploring the anti-
diabetogenic effect of mAbs directed against CD44 constant
epitopes. To this end, we resorted to the well established (7)
NOD mouse transfer model, using the same experimental
protocol we had previously adopted to arrest CD44-positive
lymphoma dissemination (8, 9) and to inhibit the development
of collagen-induced arthritis (10). Because of the consistency of
the model and the high incidence of involved animals, we
decided to use this model, rather than the spontaneous one, to
address the major question related to this study: do CD44 and its
major ligand, HA, associate functionally with autoimmune in-
sulitis leading to IDDM, and can they serve as potential thera-
peutic targets? Here we show that, in this animal model, CD44
and HA targeting by specific antibody and enzyme, respectively,
confer appreciable resistance to diabetes.
Materials and Methods
Mice. Male and female NOD mice or BALB?c mice obtained from
The Jackson Laboratory or Harlan Laboratories (Jerusalem) were
maintained in a specific pathogen-free animal facility at 21°C and
supplied with acidified water (pH 2.7) and mouse chow ad libitum.
mAbs and Enzymes. The following rat anti-mouse mAbs were used:
the anti-mouse CD44 constant region (rat, IgG2b), obtained from
hybridoma IM7.8.1 (American Type Culture Collection, TIB-235)
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: IDDM, insulin-dependent diabetes mellitus; CD44v, variant CD44; HA, hy-
aluronic acid; NOD, non-obese diabetic; IPGTT, intraperitoneal glucose tolerance test;
bHABP, biotinylated HA binding protein; MNCs, mononuclear cells; DTH, delayed type
hypersensitivity.
?Towhomreprintrequestsshouldbeaddressedat:LautenbergCenterforGeneralandTumor
Immunology, Hebrew University-Hadassah Medical School, P.O. Box 12272, Jerusalem 99120,
Israel. E-mail: naord@md2.huji.ac.il.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
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(11); the anti-mouse CD44 constant region (rat, IgG2b), obtained
from hybridoma KM81 (American Type Culture Collection, TIB-
241) (12); the anti-mouse CD44 constant region (rat, IgG2a),
obtained from hybridoma IRAWB14.4 (provided by P. W. Kin-
cade, Oklahoma Medical Research Foundation) (13). The anti-
mouse cell surface Ig idiotype (rat, IgG2b) obtained from hybrid-
oma 4D2 (provided by J. Haimovich, Tel Aviv University) (14) or
rat IgG2b Ig (PharMingen) were used as an isotype-matched
controlmAb,toexcludeFc-mediatedeffects.Thehybridomaswere
injectedintotheperitonealcavityofnudemice,andthemAbswere
purified from the ascitic fluid by protein G-Sepharose chromatog-
raphy,asdescribedinourpreviousstudy(15).Heparinase(H-2519)
and hyaluronidase (H-3757) were obtained from Sigma.
Experimental Design. Diabetic [confirmed by glucosuria (see be-
low)] female donor NOD mice, aged 15–20 weeks, were killed, and
suspensions of spleen leukocytes were washed twice in RPMI 1640
medium containing 10% fetal calf serum. A quantity of 25 ? 106
leukocytes was injected i.v. into each irradiated (550 cGy) male
recipientmouse,aged8–10weeks.Anumberofrecipientmicewere
injected with PBS whereas the remaining animals were injected
with 150 ?g of IM7.8.1 anti-CD44 or control mAbs per mouse. A
200-?l volume of PBS or mAbs (diluted in PBS) was injected i.v.
into the recipient mice 1 hr before cell transfer and then i.p. every
other day for 4 weeks (a total of 12 injections of 150 ?g of mAb per
mouse). To determine the ability of anti-CD44 mAb to inhibit
IDDM after its onset (i.e., prediabetes), NOD recipient mice were
checkedtwiceweeklyforthedevelopmentofprediabeticactivityby
using the intraperitoneal glucose tolerance test (IPGTT) (see
below).AnymouseexhibitingapositiveIPGTT(usually4–7weeks
after cell transfer) was randomly assigned to one of two groups
given IM7.8.1 anti-CD44 mAb or isotype-matched control mAb
(4D2), according to the above protocol. To monitor the develop-
ment of diabetes, the percentage of disease-free animals (glucos-
uria negative) was recorded over time. Each experiment was
repeated at least two or three times. The statistical significance of
the findings was determined (unless otherwise indicated) by the
GeneralizedGehantest(Breslow)(16).Toavoidpresentationbias,
repeated experiments were pooled (unless otherwise indicated),
and the statistical analysis was adjusted to account for interexperi-
mental variations (16).
Assessment of Diabetes by Glucose Determination. Diabetes was
monitored by testing urinary glucose with a Teststrip (Medi-Test,
Combi9,Macherey-Nagel,Du ¨ren,Germany)twiceweeklyandwas
considered positive after the appearance of glucosuria in at least
two determinations. The IPGTT was performed as follows: blood
was drawn from the paraorbital plexus at 0 min and 60 min after an
i.p. injection of glucose (1 g?kg body weight). Plasma glucose levels
were determined (as glucose mmol?liter) with a Glucose Analyzer
2 (Beckman Instruments). A glucose level above 15 mmol?liter at
the 60 min time point was considered a positive IPGTT.
Histopathology. Pancreatic tissue was fixed in 10% buffered forma-
lin and was embedded in paraffin, and 5-?m sections were stained
with hematoxylin and eosin. Sections containing a total of more
than 17 islets from each pancreas were screened and scored by an
uninformed observer according to the following criteria: 0, no cell
infiltration; 1, periinsulitis and cell infiltration involving up to 20%
of islet area; 2, cell-infiltration involving up to 50% of islet area; 3,
cell infiltration involving up to 75% of islet area; and 4, cell
infiltration involving 90–100% of islet area.
CD44 and HA Histochemistry. CD44 and HA histochemistry of pan-
creatic sections were performed as described in ref. 17, except that
normal rabbit, rather than goat, serum and rabbit, rather than goat,
anti-rat IgG secondary antibody were used. The slides were coun-
terstained with hematoxylin and were mounted with permount.
Delayed Type Hypersensitivity (DTH) Assessments. Solutions of 2%
and 0.5% 4-ethoxymethylene-2-phenyl-oxazolin-5-one (oxazolone,
E-0753, Sigma) were prepared by dissolving 200 and 50 mg,
respectively, of oxazolone in 8 ml of acetone and 2 ml of mineral oil
(Kodak). The abdomen of each mouse was painted with 100 ?l of
2% oxazolone. On day 6, the right ear was painted with 10 ?l of
0.5% oxazolone, and differences (?) between right and left ear
thickness, indicating DTH development, were determined by mi-
crocaliper.
Results
CD44 and HA Histochemical Analysis of Diabetic Pancreatic Islets.
Diabetes was induced in sublethally irradiated male NOD mice by
transfer of splenocytes isolated from diabetic female NOD mice.
After 20–50 days, the recipient mice developed diabetes, as indi-
cated by the appearance of glucose in the urine. Immunohisto-
chemical analysis of pancreatic sections prepared from the diabetic
recipient mice revealed that mononuclear cells (MNCs), mostly
lymphocytes,infiltratingtheisletsofLangerhans,aswellastheislet
cells themselves, were intensively stained after incubation with
KM81 (Fig. 1 A and B) or IM7.8.1 (results not shown) anti-CD44
mAbsandtheindicatorantibody.Similarsectionsdidnotreactwith
the same concentrations of rat isotype-matched (IgG2b) Ig (Fig.
1C) or rat isotype-matched irrelevant mAb (4D2) (results not
shown). The pancreatic islets of normal BALB?c mice failed to be
stained by the same concentration of the diabetic islet-interacting
anti-CD44 mAb and the indicator antibody (Fig. 1D). An entirely
different reaction pattern was revealed with 9A4 anti-CD44v6 and
10D1anti-CD44v4mAbs(providedbyJ.Moll,KarlsruheResearch
Center, Karlsruhe, Germany). The anti-CD44v6 mAb intensively
stained small groups of epithelial islet cells, but not the infiltrating
MNCs. Identical concentrations of anti-CD44v4 mAb, displaying
the same isotype (rat, IgG1), did not stain the islets of diabetic
recipientmice.Neitherofthetwoantibodiesstainedthepancreatic
islets of BALB?c mice (results not shown).
TheHAcontentofthediabeticisletsofrecipientNODmicewas
analyzed with biotinylated HA binding protein (bHABP) (17) and
theindicatorstreptavidin-alkalinephosphatase.Hyaluronatestain-
ing was detected along the connective tissue surrounding the islets
(Fig. 2A) and in a few small blood vessels penetrating the islets (not
shown in this figure). HA staining was prevented by preincubating
the pancreatic sections with hyaluronidase (17) (Fig. 2B), proving
thespecificityofthereaction.Incontrast,HAwasbarelydetectable
after staining with bHABP in sections of pancreatic islets derived
from normal BALB?c mice (Fig. 2C), suggesting that the inflam-
matory cascade induces the expression of hyaluronate.
The above results imply that both CD44 and HA are up-
regulated in the pancreatic islets of the diabetic recipient NOD
mice, suggesting that cell surface CD44 and matrix HA support the
migration of destructive inflammatory cells into the islets. There-
fore, we tested the ability of anti-CD44 mAb and the enzyme
hyaluronidase to interfere with this event, thereby protecting the
insulin-producing ? cells and, consequently, sustaining glucose
homeostasis.
The Anti-Diabetogenic Effect of Anti-CD44 mAb. The anti-
diabetogenic effect of the anti-CD44 mAb, which recognizes a
constant epitope on the CD44 receptor, was tested in recipient-
irradiated male NOD mice infused with splenocytes of female
diabetic NOD mice. The data pooled from 10 similar experiments
are presented in Fig. 3. The recipient mice were injected with PBS,
anti-CD44 mAbs, or control mAb, using the protocol described in
Materials and Methods. Diabetes development over time was mon-
itored by recording the appearance of glucose in the urine. Within
55 days, 90% of the recipient mice treated with PBS or with
isotype-matched (IgG2b) control mAb (4D2) developed diabetes.
In contrast, within the same period, 55% of the mice injected with
IM 7.8.1 anti-CD44 mAb were diabetes-free, and close to 50%
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remained free of disease at day 75: i.e., 47 days after termination of
antibody administration. On day 100 after transfer (or 72 days after
terminationofantibodyadministration),12of46miceinjectedwith
anti-CD44 mAb (26%) did not show any signs of diabetes. At that
time, only 2 of the 93 control mice (?2%) were free of diabetes.
Similarly to the control groups, all mice injected with IRAWB14.4
anti-CD44 mAb developed diabetes within 55 days after cell
transfer, although some delay in disease appearance was observed.
To evaluate the anti-diabetogenic effect of anti-CD44 mAb on
the active disease, the standard injection protocol was delayed by
several weeks to include prediabetic recipients with positive
IPGTT.Fig.4revealsthat45%ofthemiceinjectedwithanti-CD44
mAb did not develop diabetes for at least 100 days after the
detection of prediabetes by IPGTT (or 72 days after termination of
antibody administration) whereas all mice injected with 4D2 mAb
were diabetic on day 100. This experiment demonstrates that
anti-CD44 mAb induces considerable resistance to diabetes even
when the recipients are treated at an early stage of detectable
disease.
To demonstrate the association between the inhibition of dia-
betes and blockage of islet inflammation attributable to anti-CD44
mAb treatment, we scored islet cell infiltration in experimental
recipient mice injected with this antibody and in control mice
injected with the 4D2 isotype-matched control antibody, using the
standardprotocoldescribedinFig.3.Recipientmicewerekilled56
days after cell transfer, when the anti-diabetogenic effect of anti-
CD44 antibody was revealed by the glucose test. The degree of
inflammation in each screened pancreatic islet was histopatholog-
icallyanalyzedandscoredbyanoninformedobserver(seeMaterials
and Methods and legend to Table 1). The average inflammation
Fig. 1.
pancreasderivedfromdiabeticrecipientNODmice(A,B,andC)ornormalBALB?c
mice(D)wereincubatedwithKM81anti-CD44mAb(A,B,andD)orwiththesame
concentration (5 ?g?ml) of isotype-matched control Ig (IgG2b) (C). Bound anti-
bodieswereidentifiedwithbiotinylatedrabbitanti-ratIgG(7.5?g?ml),usingthe
avidin-biotin horseradish peroxidase detection system. CD44 is present on infil-
tratingMNCsandlocalcellsofdiabetogenicislets(AandB)butnotonnormalislet
cellsofBALB?cmice(D).CD44stainingofdiabetogenicisletsisnotobservedafter
their preincubation with isotype-matched control Ig (C). (A, ?176; B–D, ?352.)
CD44 immunohistochemical analysis of pancreatic islets. Sections of
Fig. 2.
recipients(AandB)ornormalBALB?cmice(C),pretreated[100relativeturbidity
reducing units (rTRU)?ml] (B) or not pretreated (A and C) with hyaluronidase,
were incubated with bHABP (2.5 ?g?ml). Bound HA was identified by using the
avidin-biotin peroxidase detection system. HA was detected in the connective
tissue surrounding the islets (A) whereas normal islets (C) were only marginally
stained by the same concentration of bHABP. Preincubation with hyaluronidase
prevented the bHABP staining of the diabetogenic islets (B). (?352.)
HA histochemical analysis of pancreatic islets. Sections from diabetic
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score for 139 islets from four mice injected with control mAb was
2.4 whereas the average score of 156 islets from four mice injected
withanti-CD44mAbwas0.7(Table1),implying3.4?lessintensive
islet cell infiltration. Examples of histological sections of pancreatic
islets obtained from a different experiment, showing results similar
to those listed in Table 1, are presented in Fig. 5. The islets of the
control recipient mice are heavily infiltrated by MNCs (Fig. 5 A–C;
insulitis score close to 3) whereas those of the anti-CD44 mAb-
injected recipients display only a small focal infiltrate at the
periphery, especially around blood vessels (Fig. 5 D–F; insulitis
score of up to 1).
Anti-CD44 mAb Injection Does Not Influence Normal Immune Re-
sponses of Recipient NOD Mice. The finding that anti-CD44 mAb
induced appreciable resistance to diabetes in recipient NOD mice
raised the question of whether normal immunological responses
weresimultaneouslyaffectedbytheantibody.Forinvivotesting,we
chose the delayed type hypersensitivity reaction, because of its
resemblance to the inflammatory responses in autoimmune dis-
eases. Recipient NOD mice were injected with 150 ?g?mouse IM
7.8.1 anti-CD44 mAb or with the same amount of isotype-matched
control mAb (4D2), using our standard protocol. When the anti-
diabetogenic effect of anti-CD44 mAb was already evident (Fig. 6),
the DTH response to oxazolone was assessed by measuring the
difference in thickness (?) (in mm ? 10?2) between the hapten-
painted right ear and nonpainted left ear. Recipient mice given IM
7.8.1 anti-CD44 mAb had a ? of 7.5 ? 1.3 (n ? 6), and animals
injected with isotype-matched control mAb (4D2) had a ? of 5.8 ?
0.6 (n ? 7), which is statistically insignificant (P ? 0.45 by the
Mann–Whitney U test). On day 31 after cell transfer, all of the
experimental animals were killed, and the proliferative response of
their splenocytes to stimulation with Con A was measured by
3H-thymidine incorporation. In this and in an additional indepen-
dent experiment, no difference was observed in the mitogen-
induced proliferation response (results not shown). These results
were confirmed by an additional experiment in which injection of
anti-CD44 mAb induced, in contrast to control mAb (4D2), anti-
diabetogenicactivitywhereastheoxazoloneDTHresponsewasnot
influenced. The average of ? units (mm ? 10?2) in recipient mice
Fig. 3.
injected with IM7.8.1 anti-CD44 mAb, isotype-matched control mAb (4D2),
IRAWB14.4anti-CD44mAb,orPBS,1hrbeforesplenocytetransferfromdiabetic
NOD female mice. The mice were then injected with the same reagents on
alternate days for the next 4 weeks, as described in the experimental protocol.
Developmentofdiabeteswasmonitoredbyglucosuriadetermination.Thedata
pooledfrom10similarexperimentsareshown.TheBreslowstatisticalanalysis(p)
was used to compare the PBS group with all of the other groups. NS, not
significant.
The anti-diabetogenic effect of anti-CD44 mAb. Male NOD mice were
Fig. 4.
female mice with onset of active prediabetes, as determined by IPGTT 4 to 7
weeksaftercelltransfer,wererandomlyassignedtotwogroups.Onegroupwas
injected with IM7.8.1 anti-CD44 mAb, and the other with isotype-matched con-
trol mAb (4D2) on alternate days for 4 weeks. Development of diabetes was
monitored by glucosuria determination. Data were pooled from two similar
experiments. The Breslow statistical analysis (p) was used to compare the 4D2
groupwiththeIM7group.NotethattheIPGTTpositiveprediabeticmicewerestill
glucosuria negative at the beginning of the experiment. Therefore, they were
categorized as ‘‘diabetes-free mice.’’
Inhibition of prediabetes onset by anti-CD44 mAb. Recipient NOD
Table 1. Insulitis inhibition by anti-CD44 mAb
Injected mAbMouseNo. of scored islets Average score?islet
4D21
2
3
4
17
24
58
40
139
3.0
1.5
2.9
2.2
9.6 Total
Mean ? 2.40 ? 0.35
0.7
0.8
0.5
0.9
2.9
Mean ? 0.72 ? 0.08
IM7.8.15
6
7
8
40
26
51
39
156Total
Male NOD mice were injected with anti-CD44 mAb or isotype-matched
control mAb (4D2), using the protocol described in Fig. 3. Insulitis was graded
on a scale of 0–4, 56 days after cell transfer: i.e., 28 days after termination of
antibody administration, as described in Material and Methods. At this time,
theanti-diabetogeniceffectoftheanti-CD44mAbwasclearlyobservedbythe
glucose test. The number of scored islets per mouse and the average insulitis
scorepermouse,aswellasthetotalscoreandmean?SEMforeachgroup,are
shown. P ? 0.03, by the Mann–Whitney U test.
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injectedwithIM7.8.1anti-CD44mAbwas14.3?1.2(n?5)versus
16.3 ? 1.62 (n ? 7) in the control mice (P ? 0.9 by the Mann–
Whitney U test). Collectively, these findings imply that, under these
experimental conditions, the anti-diabetogenic dose of anti-CD44
mAbdidnotinfluencecellularimmuneresponsessuchasDTHand
mitogen-induced T lymphocyte proliferation.
The Anti-Diabetogenic Effect of Hyaluronidase. Because the interac-
tion between cell surface CD44 and HA may be critical to the
development of the diabetogenic inflammatory cascade, we tested
whether degradation of HA by hyaluronidase would ameliorate
disease pathology. More than 90% of recipient mice injected 1 hr
before cell transfer and then every other day for 4 weeks with PBS
or with 20 units of the control enzyme heparinase developed
diabeteswithin60days.Incontrast,recipientsinjectedwith20units
of hyaluronidase on the same days generated considerable resis-
tancetothedisease(P?0.003,hyaluronidasegroupvs.heparinase
group)(Fig.7).Notethattheheparinaseandthehyaluronidasehad
similar specific activities.
Discussion
CD44 molecules play a critical role in the development of
inflammatory responses, leading to physiological eradication of
the invading microorganisms (18) or of the pathological mani-
festations of autoimmunity (10, 19–22). We have shown here
that both the CD44 receptor and HA, its principal ligand, are
involved in the development of IDDM in NOD mice, as deduced
from the anti-diabetogenic effects of anti-CD44 mAb and the
enzyme hyaluronidase, respectively.
Immunohistochemical staining showed that the pancreatic islets
of diabetic NOD mice contain infiltrating lymphocytes expressing
CD44(ref.23andFig.1).Usingthereversetranscriptase–PCR(24)
or immunohistochemical analysis (Fig. 1), it further was demon-
strated that splenocytes and local cells of pancreatic islets from
diabeticNODmice(butnotfromBALB?cmice)displayenhanced
expression of CD44 isoforms, including CD44 isoform(s) contain-
ing v6 epitopes. Furthermore, we showed by histochemical analysis
(Fig. 2) that, in contrast to normal pancreatic islets, the islet
periphery of a diabetogenic pancreas, as well as its endothelium,
contain HA, the principal ligand of CD44. These findings suggest
that the inflammatory cascade in the diabetogenic pancreas is
associated with the up-regulation of both CD44 and hyaluronan.
Indeed, it was demonstrated that the proinflammatory cytokines
tumor necrosis factor-? and IL-1?, as well as bacterial lipopolysac-
charide, induce expression of HA in endothelial cell lines and on
Fig. 5.
(A–C) and from the corresponding recipients treated with anti-CD44 mAb (D–F).
Pancreatictissuefromrecipientmice(asdescribedinthelegendtoTable1)were
fixed in 10% buffered formalin, embedded in paraffin, and 5-?m sections were
stainedwithhematoxylinandeosin.Thepancreaticendocrineisletsofthecontrol
recipients are heavily infiltrated by MNCs (A–C) whereas those of the anti-CD44
mAb-injected mice display a small focal infiltrate at the periphery (D–F). (?158.)
Histological analysis of pancreatic islets derived from control recipients
Fig. 6.
development. Male NOD mice were injected with IM7.8.1 anti-CD44 mAb or
isotype-matchedcontrolmAb(4D2)1hrbeforesplenocytetransferfromdiabetic
femaleNODmice,andtheneveryotherdayforanadditional4weeks.Onday20
aftercelltransfer,whentheanti-CD44mAbanti-diabetogeniceffectwasevident,
all recipients were sensitized with oxazolone, which was applied to their abdo-
men.After6days,theywererepaintedintherightear.Thedifferenceinthickness
betweentherightandtheleftear(?)wasdeterminedbymicrocaliper1daylater.
Developmentofdiabeteswasmonitoredbyglucosuriadetermination.Statistical
analysis was carried out as described in Fig. 4. The difference in DTH (? ear
thickness) between the two groups is statistically insignificant according to the
Mann–Whitney U test.
The anti-diabetogenic dose of anti-CD44 mAb does not influence DTH
Fig. 7.
wereinjected1hrbeforecelltransferandtheneveryotherdayfor4weekswith
PBSor20unitsofheparinase(Hep’ase)orhyaluronidase(H’ase).Developmentof
diabetes was monitored by glucosuria. Data were pooled from two similar
experiments.
The anti-diabetogenic effect of hyaluronidase. Recipient NOD mice
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