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*
Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305;
Department of Veterans Affairs, Palo Alto Health Care System, Palo Alto, CA 94304; and
DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nonobese diabetic (NOD) mice are a well-known model for human IDDM. The key role of T cells in this diabetes model is indicated by the predominance of T cells in the inflamed islets, the prevention of diabetes by neonatal thymectomy, the absence of diabetes in NOD/scid mice, and the ability of splenic T cells from diabetic NOD mice to transfer insulitis and diabetes (5, 6, 7, 8). NOD mice also develop lymphocytic inflammation in submandibular salivary glands (sialoadenitis) and lacrimal glands (dacryoadenitis) (9, 10). Thus, NOD mice serve as a model for human Sjogren’s syndrome, an autoimmune disease characterized by inflammation and destruction of salivary and lacrimal gland tissue followed by development of dry mouth and eyes (11). Splenic T cells from old NOD mice can transfer salivary gland inflammation to young NOD or NOD/scid mice, indicating that T cells are involved in the development of this inflammation (6, 12). Thus, migration of autoreactive and effector T lymphocytes from blood into extranodal tissues is a key event in the pathogenesis of pancreatic islet and salivary gland inflammation, and in the development of diabetes.
Lymphocyte migration from blood into tissue involves a complex adhesion cascade with sequential lymphocyte/endothelial adhesion and activation steps (reviewed in Refs. 13, 14, 15). Adhesive interactions of lymphocytes under flow to PP or peripheral LN (PLN) high endothelial venules (HEV) consist of at least four steps: 1) an initial transient tethering and rolling; 2) if the lymphocytes encounter appropriate activating factors such as chemokines in the local environment, rolling may be followed by a lymphocyte activation step mediated primarily through G protein-linked chemoattractant receptors, which then leads to; 3) firm adhesion or sticking mediated by activated integrins, which may be followed by; 4) lymphocyte diapedesis into tissue (16, 17, 18). Interference with any of these steps would be expected to inhibit lymphocyte migration into tissue.
CD43 (leukosialin, sialophorin) is a large sialoglycoprotein that is expressed by most hematolymphoid cells including T cells, but not (or only at very low levels) by mature conventional B (B-2) cells (19, 20). CD43 has been implicated in a variety of adhesive and anti-adhesive events in the immune system (21, 22, 23, 24, 25, 26, 27, 28). We have developed and characterized a unique anti-mouse CD43 mAb, known as L11, that is a potent inhibitor of T cell migration from the blood into organized lymphoid tissues including LN, PP, and spleen (26). Our in situ studies indicate that L11 blocks the activation-dependent arrest and firm adhesion of T cells in PP HEV (L.M.M., unpublished observation). These results led us to hypothesize that L11 might effectively interfere with T lymphocyte migration into extranodal sites of chronic inflammation and thus might be a useful immunotherapeutic agent for the treatment of T cell-mediated autoimmune diseases such as IDDM.
In the current study, we asked if L11 could block the migration of T cells from the bloodstream into extranodal sites of inflammation. Using a sensitive lymphocyte transfer technique (29, 30), we demonstrated that L11 significantly inhibits the migration of T cells into inflamed pancreatic islets and submandibular salivary glands of NOD mice. Next, we asked whether L11 is an effective immunotherapeutic agent for the prevention of autoimmune diseases. We showed that L11 treatment of prediabetic NOD mice significantly inhibits the development of diabetes. This protection is long-lived, with decreased incidence of diabetes even months after cessation of Ab administration. Moreover, if the treatment was started before the onset of inflammation, L11 also inhibits the development of inflammation in salivary gland and pancreatic islets.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NOD mice were obtained from our colony or from Taconic Farms
(Germantown, NY). The incidence of diabetes in female mice from both
groups is 
80% by 30 wk of age. NOD/LtSz-scid/J (hereafter referred
to as NOD/scid) and NOD.NON-Thy1a mice were bred
in our colony from stock obtained from The Jackson Laboratory (Bar
Harbor, ME). Female mice were used in all experiments.
Monoclonal Abs
L11 (anti-mouse CD43; rat IgG2a) and species- and isotype-matched negative control mAbs Hermes-1 (anti-human CD44), 53-2.1 (anti-mouse Thy-1.2; provided by Dr. J. Ledbetter, Seattle, WA), and M1/9 (anti-mouse CD45; American Type Culture Collection (ATCC), Manassas, VA) were used for in vivo experiments. The negative control mAbs do not block lymphocyte migration to organized lymphoid tissues and do not affect the development of sialoadenitis, insulitis, or diabetes (31, 32) (data not shown). These mAbs were grown as ascites in C57/scid or outbred nude mice and purified by protein G chromatography using endotoxin-free reagents as described (33).
Monoclonal Abs used for flow cytometric analysis or immunohistochemical
staining included anti-CD43 (mAb L11), anti-Thy-1.2 (53-2.1),
anti-CD3 (145-2C11; ATCC), anti-CD4 (GK1.5; ATCC), anti-CD8
(53-6.72; ATCC), anti-B220 (RA3-6B2; provided by Dr. R. Coffman,
Palo Alto, CA), anti-CD45 (M1/89; ATCC),
anti-
4 integrin (R1-2; ATCC),
anti-ß7 integrin (Fib-504),
anti-4ß7 heterodimer (DATK-32),
anti-L-selectin (MEL-14; ATCC), anti-CD44 (IM7; ATCC),
anti-LFA-1 (FD441.8; ATCC), anti-PNAd (MECA-79),
anti-MAdCAM-1 (MECA-367), anti-VCAM-1 (M/K-2.7; ATCC),
anti-ICAM-1 (YN1/1.7; ATCC), and anti-macrophage (F4/80,
ATCC).
In vivo T cell migration assay
The ability of L11 to block the migration of T cells from blood
into extranodal inflammatory sites was evaluated using short-term in
vivo migration assays in Thy-1 congenic mice (6, 29, 30).
Briefly, cell suspensions from PLN, mesenteric LN (MLN), and red
cell-lysed spleen of 12- to 24-wk-old prediabetic NOD (Thy-1.2) mice
were incubated on ice for 15 min with 20 µg/ml L11, 20 µg/ml M1/9
(negative control mAb), or media. Suspensions were washed, and 7
x 107 cells were injected i.v. into prediabetic
female NOD.NON-Thy1a (Thy-1.1) mice. The ages of
the host mice are indicated in Table I
.
In some experiments, NOD.NON-Thy1a mice were
given PBS or 7 x 107 syngeneic spleen
cells, instead of congenic spleen cells. Host mice were sacrificed
2 h after injection; this time point allows us to evaluate the
primary migration of lymphocytes from blood into tissue, without
significant effects of recirculation or Ag-specific retention. Spleen,
MLN, pancreas, and salivary and lacrimal glands were removed and frozen
in OCT compound (Miles, Elkhart, IN). Hematoxylin- and eosin-stained
sections of the lacrimal glands did not show any inflammation; these
tissues were not analyzed further. Frozen sections of the other tissues
were stained using a two-stage avidin biotin system. Briefly, the
sections were incubated with biotin-conjugated 53-2.1, which reacts
with Thy-1.2 (donor T cells) but not Thy-1.1 (host T cells), or with
biotin-conjugated 6B2 (B cells), GK1.5 (CD4), 145-2C11 (CD3), or
Hermes-1 (negative control). Following two washes in PBS, sections were
sequentially incubated with peroxidase-streptavidin (Zymed, South San
Francisco, CA), diaminobenzidine/hydrogen peroxide, and methylene blue
counterstain. Donor T cells (stained with mAb 53-2.1) were identified
and counted in inflamed foci in pancreatic islets, in T cell zones in
inflamed salivary gland, and in T cell zones in spleen (periarteriolar
sheath) and MLN (paracortex). The T cell zones were identified by
examining adjacent sections stained with mAb against CD4 or CD3.
Surface areas of the T cell zones were determined using an image
analysis system (VAS II; Mideo Systems, Huntington Beach, CA)
(29). In inflamed islets, there were few B cells and no
discrete B cell follicles, so the entire surface area of inflammation
was determined. A minimum area of 0.1 mm2 was
evaluated in each tissue from each mouse. The number of donor T
cells/mm2 in each microenvironment was
calculated.
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In vivo immunotherapy
NOD mice from different litters were randomized into treatment groups under one of the following protocols: 1) mice were given mAb (40 µg mAb/g body weight) or PBS i.p. every third day from 1 to 4 wk of age, or; 2) mice were treated with mAb by s.c. osmotic pump (Alza, Palo Alto, CA; Alzet model 2004; 75 µg/mAb or 150 µg/mAb per day) from 8 to 12 wk of age. In preliminary experiments, these doses resulted in detectable levels of L11 and negative control mAbs (M1/9, 53-2.1) on peripheral blood T cells throughout the treatment (data not shown). Mice were monitored for development of diabetes or sacrificed for collection of tissues as described below.
Determination of diabetes incidence
NOD mice were tested for glycosuria twice weekly beginning at 12 wk of age. When a mouse had two positive urine tests, its blood glucose level was determined; if >13.9 mmol/L (250 mg/dl), the mouse was considered to be diabetic and was sacrificed. The date of diabetes onset was the day on which glycosuria was first detected.
Tissue collection and analysis
Mice from the in vivo immunotherapy experiments, sacrificed when
diabetic or at times indicated in the text, donated some of the
following tissues: 1) Peripheral blood was taken for leukocyte
count and differential. 2) Suspensions of PLN, MLN, spleen, and thymus
were stained using two- and three-color immunofluorescence (IF)
protocols and analyzed on a FACScan or FACScaliber flow cytometer
(Becton Dickinson) as described (34). Data are presented
as percentage of cells reacting with a specific mAb minus percentage of
cells reacting with negative control mAb, or as absolute number of
cells reacting with a specific mAb. 3) Pancreas, submandibular salivary
gland, lacrimal gland, MLN, and, in some mice, PLN and spleen were
collected and fixed in formalin for routine histology or frozen in OCT
compound for immunohistology. For immunohistologic evaluation, frozen
sections were stained with a three-stage immunoperoxidase technique and
evaluated as described (30). To determine the degree of
inflammation, hematoxylin- and eosin-stained sections of frozen or
formalin-fixed pancreas and salivary gland and lacrimal gland were
scored as follows: pancreas: 0, no inflammation; 1, few lymphocytes
next to the islets (mild peri-insulitis); 2, many lymphocytes next to
the islets (marked peri-insulitis); 3, lymphocytes infiltrating the
islets (insulitis); salivary and lacrimal gland: 0, no inflammation; 1,
small foci of perivascular/periductular inflammation; 2, large foci of
perivascular/periductular inflammation; 3, large foci of inflammation
not confined to perivascular/periductular areas (35). 4)
Spleens were taken from 13-wk-old NOD mice that had been treated with
150 µg/day of L11 or M1/9, or PBS, from 8 to 12 wk of age as
described above. One million cells were stimulated in vitro in
triplicate in wells coated with anti-CD3 (mAb 145-2C11, 5 µg/ml).
The culture supernatants were collected after 48 h, and the
concentrations of IL-4 and IFN-
were determined using ELISA as
recommended by the manufacturer (Endogen, Woburn, MA). 5) Twelve-wk-old
mice treated with mAbs or PBS from 1 to 4 wk of age were evaluated for
the ability to develop a cutaneous delayed-type hypersensitivity
response, as described (36). Briefly, abdominal skin was
painted with 25 µl of 2,4-dinitrofluorobenzene (DNFB) in 4:1
acetone/olive oil vehicle on days 0 and 1. On day 5, mice were
challenged on one ear with 20 µl of 0.2% DNFB while the
contralateral ear was treated with vehicle only. Ear thickness was
measured before challenge and at 24 and 48 h after challenge using
a Fowler caliper. Values are presented as change in thickness from the
prechallenge measurement.
Adoptive transfer
In experiments to detect cells that suppress the adoptive transfer of diabetes and salivary and lacrimal gland inflammation, 6- to 8-wk-old NOD/scid mice received i.v. injections of: 1) 2 x 107 cells obtained from a pool of splenocytes prepared from recently diabetic NOD mice; or 2) 2 x 107 cells obtained from a pool of splenocytes prepared from recently diabetic NOD mice and 2 x 107 splenocytes from 20-wk-old prediabetic NOD mice that had been treated from 8 to 12 wk of age with 150 µg/day of L11 or negative control mAb (M1/9) (each host received cells from a single L11 or negative control mAb-treated donor); or 3) 2 x 107 splenocytes from 20-wk-old prediabetic NOD mice that had been treated with L11 or negative control mAb; none of the mice in this group developed diabetes by 70 days after transfer. The host mice were followed for the development of diabetes. Diabetic mice were sacrificed, and salivary and lacrimal glands were collected for histologic examination, as described above.
Statistics
Fisher’s exact test was used to analyze differences in the incidence of diabetes between treatment groups. Mann-Whitney U test (two-tailed) was applied to evaluate differences in tissue inflammation scores and in numbers of T and B cells. Student’s t test was used to evaluate cytokine concentrations and ear swelling.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the ability of L11 to inhibit lymphocyte migration from
the bloodstream into extranodal sites of inflammation, spleen and LN
lymphocytes from NOD (Thy-1.2+) mice were
transferred i.v. into congenic NOD.NON-Thy1a
(Thy-1.1+) host mice. The donors were old (>12
wk) prediabetic female mice, as their spleens are enriched in memory
cells that should be able to home to different sites of inflammation
(2, 37). Host mice were sacrificed 2 h after
injection; the localization of donor T cells to host MLN, spleen,
pancreas, and salivary gland was determined using tissue-section
immunohistochemical staining with an allotype-specific anti-Thy-1.2
mAb (53-2.1). Large numbers of donor T cells were found in the spleen
and MLN of host mice that received cells treated with media or with a
negative control mAb (Table I
; Fig. 1);
most of these cells were in T cell areas (spleen periarteriolar sheath
(Fig. 1) and MLN paracortex). Smaller numbers of donor T cells were
found in inflamed foci in pancreas and salivary gland (14 and 20%,
respectively, of that in MLN paracortex; mean for negative control
mAb-treated cells, Table I, Expts. 1 and 2). The inflamed areas in the
salivary glands had T and B cell zones, as determined by staining with
mAb against B220, CD4, and CD3; the donor T cells were found mainly in
the T cell zones (Fig. 1). In inflamed pancreatic islets, the host
infiltrate was mostly T cell, with few B cells and no discrete B cell
follicles; the donor T cells were scattered throughout the infiltrate
(Fig. 1). Monoclonal Ab 53-2.1 did not stain lymphocytes in sections of
spleen, MLN, pancreas, or salivary gland of
NOD.NON-Thy1a mice that had received PBS or
syngeneic spleen cells (data not shown). As expected, L11 blocked the
migration of T cells from the bloodstream into MLN and spleen (Table I;
Fig. 1) (26). Moreover, there was significant inhibition
of T cell migration to inflamed islets and inflamed salivary gland
(Fig. 1; Table I: mean inhibition in two experiments, 83 and 90%,
respectively).
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Immunotherapy with anti-CD43 mAb L11 prevents the development of diabetes
To determine the ability of L11 to prevent the spontaneous
development of diabetes, female NOD mice were treated with L11, with
isotype- and species-matched negative control mAb, or with PBS. Greater
than 75% of mice treated from 1 to 4 wk of age with negative control
mAb or PBS developed diabetes by 52 wk of age. In contrast, none of the
eight mice treated with L11 became diabetic (Fig. 2A) (L11 vs negative control
mAb, p = 0.0011; L11 vs PBS, p =
0.0070; Fisher’s exact test). If treatment was started when the mice
were 8 wk old and already had some insulitis (38), L11
(150 µg/mAb per day) provided significant protection against the
development of diabetes, while a lower dose (75 µg/mAb/day) of L11
was less effective (incidence of diabetes at 40 wk of age, 11 and 56%,
respectively) (Fig. 2B). In contrast, >70% of mice treated
with negative control mAbs developed diabetes by 40 wk of age (L11 150
µg/day vs negative control mAb (Hermes-1), p =
0.0198; L11 150 µg/day vs negative control mAb (53-2.1),
p = 0.0087; Fisher’s exact test).
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We asked if treatment with L11 could decrease the accumulation of
lymphocytes in and around pancreatic islets and in salivary glands. In
the first experiment, histologic sections were prepared of pancreata
and salivary glands from 18-wk-old female NOD mice that had been
treated with L11, negative control mAb (M1/9), or PBS from 1 to 4 wk of
age. The pancreata of mice treated with the negative control mAb or PBS
had extensive inflammation, with most islets showing marked
peri-insulitis (inflammation score 2) or insulitis (inflammation score
3) (inflammation score, mean ± SD: negative control mAb: 2.1
± 0.6; PBS: 2.3 ± 0.5; n = 4 mice/group) (Fig. 3A). In contrast, there was
very little inflammation in the islets of L11-treated mice
(inflammation score, mean ± SD: 0.1 ± 0.1;
n = 4 mice per group; p = 0.0286, L11
vs negative control mAb; p = 0.0286, L11 vs PBS;
Mann-Whitney) (Fig. 3A). Besides decreasing the islet
inflammation, L11 treatment partially inhibited salivary gland
inflammation (Fig. 3A) (p = 0.0571,
L11 vs negative control mAb; p = 0.1143, L11 vs PBS;
Mann-Whitney).
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B). Immunohistologic examination of the pancreata
showed no differences between groups in adhesion molecule expression by
lymphocytes (4, ß7,
LFA-1, L-selectin) or endothelial cells (MAdCAM-1, PNAd, VCAM-1,
ICAM-1). Effects of L11 immunotherapy on the peripheral immune system
We conducted several experiments to evaluate the long-term effects of L11 immunotherapy on T cell numbers, phenotypes, and functions. All experiments were performed after therapy had ended; L11 and the negative control mAb (both of which are rat IgG) were no longer detectable on the surfaces of the T cells, as indicated by lack of staining of these cells with PE-anti-rat IgG.
To determine whether L11 treatment causes permanent changes in numbers
of lymphocytes or in their expression of subset markers or adhesion
molecules, lymphoid tissues of 14-wk-old mice treated from 1 to 4 wk or
8 to 12 wk of age with L11, negative control mAb, or PBS were
suspended, stained using two- or three-color IF techniques, and
analyzed by flow cytometry. There were no significant differences
between treatment groups in total number of cells in PLN, MLN, or
thymus, or in splenic weights (data not shown). In addition, there were
no significant differences in the absolute numbers of PLN (Fig. 4) or MLN cells, or relative numbers
of spleen cells, expressing Thy-1, CD4, CD8, or B220. MLN T cells from
the different groups showed similar levels of expression of
4, ß7, L-selectin, and
CD43. Thymi from different groups showed no significant differences in
numbers of double negative (CD4-
CD8-), double positive, and single positive
thymocytes. Immunohistochemical staining of MLN showed normal patterns
of expression of lymphocyte and endothelial Ags including CD4, CD8,
CD3, B220, F4/80, L-selectin, 4,
ß7, CD43, MAdCAM-1, PNAd, VCAM-1, and ICAM-1
(data not shown). There were no significant differences in peripheral
blood leukocyte counts or differentials (percentage of lymphocytes,
monocytes, neutrophils, and eosinophils) between the treatment groups
(data not shown).
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We isolated spleen cells from 13-wk-old NOD mice that were treated from
8 to 12 wk of age with L11, negative control mAb or PBS. To determine
whether our treatment protocols altered the Th1/Th2 balance, the
splenocytes were stimulated in vitro with an activating anti-CD3
mAb and their production of IL-4 and IFN- was determined.
Splenocytes from all mice showed production of IFN-, with no
significant difference between treatment groups (L11: 104 ± 71;
negative control mAb (M1/9): 70 ± 53; PBS: 137 ± 126;
mean ± SD, ng/ml; p = 0.41, L11 vs negative
control mAb; p = 0.63, L11 vs PBS; t test,
two-tailed). There was no detectable production of IL-4.
Splenocytes from L11-treated NOD mice do not suppress the adoptive transfer of diabetes or salivary and lacrimal gland inflammation
To determine whether L11 treatment causes the induction of
suppressor cells, we asked if splenocytes from L11-treated NOD mice
could block the ability of splenocytes from diabetic NOD mice to
transfer disease into NOD/scid mice (6). Host NOD/scid
mice were given spleen cells from unmanipulated diabetic NOD mice alone
or mixed with spleen cells from 20-wk-old NOD mice that had been
treated from 8 to 12 wk of age with L11 or with a negative control mAb
(M1/9). As shown in Table II, splenocytes
from L11-treated mice did not suppress the adoptive transfer of
diabetes or salivary and lacrimal inflammation. Thus, L11 treatment of
NOD mice does not produce immunoregulatory cells that actively suppress
the adoptive transfer of disease.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD43 is a large, negatively charged sialoglycoprotein that extends 45 nm from the leukocyte surface (39). Because of its extended structure and negative charge, CD43 is thought to act as a passive barrier to the engagement of surface adhesion molecules. Evidence for this anti-adhesive role includes: 1) transfection of CD43 into HeLa cells results in decreased ability to bind T cells (21); and 2) CD43 knockout mice show enhanced T lymphocyte/endothelial adhesion and T lymphocyte migration (22). In contrast, CD43 may also have proadhesive functions. For example, stimulatory anti-CD43 mAbs can cause increased leukocyte adhesion. This adhesion is thought to be mediated through intracellular signaling that leads to increased avidity of integrins such as LFA-1 (23, 24, 25).
However, we recently showed that anti-CD43 mAb L11 inhibits T cell binding to HEV in PP and PLN and blocks T cell migration to PP, LN, and spleen (26). L11 only inhibits this binding and migration if there is cross-linking of CD43 (our unpublished observations). In addition, in situ microscopy studies of T cell interactions with PP HEV indicate that L11 has no effect on initial tethering and rolling (step 1 of the adhesion cascade) but almost completely blocks activation-dependent firm adhesion (step 3) (unpublished observation). These data suggest that L11 affects the activation and signaling pathways within the T cells; studies are currently underway to define these pathways.
There are several mechanisms by which L11 immunotherapy might prevent the development of diabetes and inflammation in NOD mice. First, the decrease in diabetes incidence and, when mice were treated from 1 to 4 wk of age, the decrease in pancreatic islet and salivary gland inflammation, may be due to direct inhibition of migration of Ag-specific and effector T lymphocytes into these tissues. This hypothesis is supported by our data showing that L11 blocks the migration of T cells from blood into inflamed pancreatic islets and salivary glands in NOD mice. L11 treatment from 8 to 12 wk of age suppressed the development of diabetes without decreasing the degree of inflammation; this could be mediated either by preventing maintenance of the pathologic cell recruitment or by additional mechanisms.
L11 may also inhibit the migration of other cell types. IDDM is generally considered to be a T cell-mediated disease; however, other leukocytes, such as monocytes, are thought to be involved in the development of insulitis and the destruction of ß cells (40, 41). CD43 is expressed by monocytes, and L11 may be able to block the migration of monocytes and perhaps dendritic cells to organized lymphoid tissues or inflamed extranodal tissues. Indeed, L11 can inhibit the binding of monocytoid WEHI cells to HEV in inflamed lymph nodes (42). Further studies would be needed to determine whether L11 blocks the migration of monocytes into inflamed pancreatic islets and whether this blocking is involved in the prevention of insulitis and diabetes.
CD43 is also involved in diverse homotypic and heterotypic adhesion events in the immune system (21, 22, 23, 24, 25, 26, 27, 28). Besides inhibiting T lymphocyte/endothelial binding, L11 might also interfere with the adhesion of T cells to other leukocytes, including APCs (43). Thus, immunotherapy with L11 might also affect the priming or activation of Ag-specific and effector lymphocytes that are involved in the development of inflammation in pancreas and salivary glands; this effect on priming and activation could be mediated by L11 blocking T cell migration to lymphoid tissues, or by L11 interfering with cell-cell interactions within these tissues. However, it is unlikely that the prevention of diabetes in our mice is simply due to lack of priming or activation of lymphocytes because L11 treatment is effective when given before (1–4 wk of age) or after (8–12 wk of age) initial detection of the anti-islet autoimmune response in NOD mice (4). In addition, we have shown that L11 inhibits the ability of splenocytes from diabetic NOD mice to transfer pancreatic islet inflammation and diabetes into young NOD/scid mice (44). Because the donor splenocytes contain T cells that are specific for islet autoantigens, the prevention of diabetes and insulitis in this adoptive transfer model could not be due to blocking of T cell priming alone (4).
The immune response leading to overt diabetes in NOD mice is of the Th1
type, with increased production of IFN- and IL-2 (45, 46). Several studies indicate that the deviation of the
autoimmune response toward a Th2 response, characterized by production
of IL-4 and IL-10, can have a protective effect on the development of
diabetes (reviewed in Ref. 46). Although we cannot exclude
L11-mediated changes in the cytokine milieu in the inflamed islets, our
results show no changes in splenocyte production of IFN- and IL-4
after immunotherapy with L11. Thus, it seems that the mechanism of
action of L11 treatment does not include generation of a Th2 cell
response that is detectable within the spleen. This is supported by our
finding that spleen cells from L11-treated NOD mice did not block
adoptive transfer of diabetes by spleen cells from diabetic NOD mice.
Clearly, L11 treatment did not induce generalized T cell depletion, as
there were no long-term changes in peripheral lymphocyte counts or
phenotypes (Fig. 4) or in the ability to mount a cutaneous delayed-type
hypersensitivity response in L11-treated mice.
In summary, our results indicate that treatment of NOD mice with L11 from 1 to 4 wk or 8 to 12 wk of age is very effective in preventing the development of diabetes. It may be desirable to produce mAbs against human CD43 that, like L11 in the mouse, block T cell migration. Our results suggest that such mAbs may be potential inhibitors of pathologic inflammatory responses in diverse settings of autoimmunity.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Sara Michie, Department of Pathology, Veterans Affairs Medical Center, 3801 Miranda Avenue, 154S, Palo Alto, CA 94304. E-mail address:
3 Abbreviations used in this paper: LN, lymph node; NOD, nonobese diabetic; IDDM, insulin-dependent diabetes mellitus; PP, Peyer’s patch; PLN, peripheral LN; HEV, high endothelial venule; MLN, mesenteric lymph node; IF, immunofluorescence; DNFB, 2,4-dinitrofluorobenzene.
Received for publication May 28, 1999. Accepted for publication September 2, 1999.
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References |
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4ß7 and LFA-1 in lymphocyte homing to Peyer’s patch-HEV in situ: the multistep model confirmed and refined. Immunity 3:99.[Medline]
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  J. L. Cannon, A. Collins, P. D. Mody, D. Balachandran, K. J. Henriksen, C. E. Smith, J. Tong, B. S. Clay, S. D. Miller, and A. I. Sperling CD43 Regulates Th2 Differentiation and Inflammation J. Immunol., June 1, 2008; 180(11): 7385 - 7393. [Abstract] [Full Text] [PDF]
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 P. D. Mody, J. L. Cannon, H. S. Bandukwala, K. M. Blaine, A. B. Schilling, K. Swier, and A. I. Sperling Signaling through CD43 regulates CD4 T-cell trafficking Blood, October 15, 2007; 110(8): 2974 - 2982. [Abstract] [Full Text] [PDF]
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M. Matsumoto, A. Shigeta, Y. Furukawa, T. Tanaka, M. Miyasaka, and T. Hirata CD43 Collaborates with P-Selectin Glycoprotein Ligand-1 to Mediate E-Selectin-Dependent T Cell Migration into Inflamed Skin J. Immunol., February 15, 2007; 178(4): 2499 - 2506. [Abstract] [Full Text] [PDF]
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D. A. Carlow and H. J. Ziltener CD43 Deficiency Has No Impact in Competitive In Vivo Assays of Neutrophil or Activated T Cell Recruitment Efficiency J. Immunol., November 1, 2006; 177(9): 6450 - 6459. [Abstract] [Full Text] [PDF]
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R. C. Fuhlbrigge, S. L. King, R. Sackstein, and T. S. Kupper CD43 is a ligand for E-selectin on CLA+ human T cells Blood, February 15, 2006; 107(4): 1421 - 1426. [Abstract] [Full Text] [PDF]
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M. L. Ford, T. M. Onami, A. I. Sperling, R. Ahmed, and B. D. Evavold CD43 Modulates Severity and Onset of Experimental Autoimmune Encephalomyelitis J. Immunol., December 15, 2003; 171(12): 6527 - 6533. [Abstract] [Full Text] [PDF]
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 E. Layseca-Espinosa, G. Pedraza-Alva, J. L. Montiel, R. del Rio, N. A. Fierro, R. Gonzalez-Amaro, and Y. Rosenstein T cell aggregation induced through CD43: intracellular signals and inhibition by the immunomodulatory drug leflunomide J. Leukoc. Biol., December 1, 2003; 74(6): 1083 - 1093. [Abstract] [Full Text]
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 B. Xu, N. Wagner, L. N. Pham, V. Magno, Z. Shan, E. C. Butcher, and S. A. Michie Lymphocyte Homing to Bronchus-associated Lymphoid Tissue (BALT) Is Mediated by L-selectin/PNAd, {alpha}4{beta}1 Integrin/VCAM-1, and LFA-1 Adhesion Pathways J. Exp. Med., May 19, 2003; 197(10): 1255 - 1267. [Abstract] [Full Text] [PDF]
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T. M. Onami, L. E. Harrington, M. A. Williams, M. Galvan, C. P. Larsen, T. C. Pearson, N. Manjunath, L. G. Baum, B. D. Pearce, and R. Ahmed Dynamic Regulation of T Cell Immunity by CD43 J. Immunol., June 15, 2002; 168(12): 6022 - 6031. [Abstract] [Full Text] [PDF]
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 A. Mikulowska-Mennis, B. Xu, J. M. Berberian, and S. A. Michie Lymphocyte Migration to Inflamed Lacrimal Glands Is Mediated by Vascular Cell Adhesion Molecule-1/{alpha}4{beta}1 Integrin, Peripheral Node Addressin/L-Selectin, and Lymphocyte Function-Associated Antigen-1 Adhesion Pathways Am. J. Pathol., August 1, 2001; 159(2): 671 - 681. [Abstract] [Full Text]
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 G. Pedraza-Alva, S. Sawasdikosol, Y. C. Liu, L. B. Merida, M. E. Cruz-Munoz, F. Oceguera-Yanez, S. J. Burakoff, and Y. Rosenstein Regulation of Cbl Molecular Interactions by the Co-receptor Molecule CD43 in Human T Cells J. Biol. Chem., January 5, 2001; 276(1): 729 - 737. [Abstract] [Full Text] [PDF]
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