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(Journal of Leukocyte Biology. 2001;70:510-517.)
© 2001 by Society for Leukocyte Biology

Leukocytes infiltrating the pancreatic islets of nonobese diabetic mice are transformed into inactive exiles by combinational anti-cell adhesion therapy

Sharada Kommajosyula*, Shiva Reddy{dagger}, Kristina Nitschke{dagger}, Jagat R. Kanwar*, Muralidhar Karanam{dagger} and Geoffrey W. Krissansen*

Departments of
* Molecular Medicine and
{dagger} Pediatrics, Faculty of Medicine and Health Science, University of Auckland, Auckland, New Zealand

Correspondence: Geoffrey W. Krissansen, Associate Professor, Department of Molecular Medicine, Faculty of Medicine and Health Science, University of Auckland, Auckland, New Zealand. E-mail: gw.krissansen{at}auckland.ac.nz Received October 6, 2000; revised March 6, 2001; revised May 25, 2001; accepted May 29, 2001.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leukocytes infiltrate the pancreatic islets of nonobese diabetic mice, causing ß-cell destruction and autoimmune Type I diabetes. Here, we completely blocked adoptive transfer of diabetes and reduced spontaneous disease incidence from 71% to 17% by simultaneously administering a combination of antibodies directed against {alpha}4, ß2, and ß7 integrins and their ligands VCAM-1, MAdCAM-1, and ICAM-1 for 52 and 28 days, respectively. CD4 and CD8 T cells and macrophages were excluded from islets and remained entrapped in a peri-islet location as inactive exiles, no longer expressing normal levels of interferon-{gamma}, interleukin-4, and iNOS. Only IL-10 expression was retained, which could aid immunosuppression. Infiltrating leukocytes retained a peri-islet location, even 215 days following suspension of antibody treatment, potentially forming a barrier to the entry of active, autoantigen-reactive T cells. Combination treatment was effective against spontaneous disease when administered from 7 days of age but ineffective when initiated late in the prediabetic period (day 40 or 70). Nevertheless, anti-{alpha}4 subunit mAb monotherapy alone was very effective, reducing insulitis to levels similar to those obtained with combinational antibody treatment, suggesting that {alpha}4 integrins are major receptors contributing to leukocyte infiltration. Treatment with anti-{alpha}4 integrin antibody retained some therapeutic benefit when administered from days 7, 40, or 70 of age. The results have implications for the treatment of diabetes and provide a unique insight into the fate of disease-forming leukocytes following anti-CAM therapy.

Key Words: integrins • MAdCAM-1 • VCAM-1 • ICAM-1 • diabetes • insulitis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Insulin-dependent diabetes mellitus (IDDM) is a multifactorial autoimmune disease, characterized by an infiltration of pancreatic islets by mononuclear cells [1 ]. All the major CD4/CD8 T-, B-, macrophage-, and dendritic-cell subsets infiltrate and initiate an immune response that leads to the selective destruction of insulin-secreting ß cells comprising most of the islet. The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes and is regarded as a suitable model of human IDDM [2 ]. As in humans, a complex mixture of mononuclear cells infiltrates the islets of NOD mice from 3 weeks of age, leading to the gradual loss of ß cells [3 4 5 ]. IDDM can be adoptively transferred by splenic T cells from diabetic donors, and both CD4 and CD8 T cells contribute to the transfer of IDDM [6 , 7 ].

The trafficking of mononuclear cells to sites of inflammation is a multi-step process involving selectins, chemoattractants, and members of the large family of heterodimeric {alpha}ß integrins [8 , 9 ]. Subsets of {alpha}4, ß2, ß7, and {alpha}V integrins mediate leukocyte motility, the firm adherence of leukocytes to vascular endothelium and/or transendothelial migration. The integrin {alpha}4ß7, which mediates the trafficking of T lymphocytes to sites of chronic inflammation [9 10 11 12 13 ], is expressed on most infiltrating cells at all stages of insulitis [14 15 16 ]. Mucosa-associated (ß7-integrinhigh) lymphocytes accumulate early in the pancreas of NOD mice and are believed to be involved in the early phases of islet inflammation [16 ]. The {alpha}4 integrin-ligand mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) is the predominant addressin expressed on endothelium next to islets during the early stages of insulitis [14 , 17 ]. Another {alpha}4 integrin ligand, namely vascular addressin cell adhesion molecule-1 (VCAM-1), is up-regulated on the vascular endothelium within inflamed islets.

Treatment of NOD mice with monoclonal antibodies (mAbs) directed against the integrin {alpha}4 subunit and VCAM-1 significantly inhibits insulitis and prevents diabetes but does not affect the immune response against a panel of pancreatic ß-cell autoantigens [15 , 18 , 19 ]. Anti-{alpha}4 mAbs were more effective at blocking leukocyte infiltration than anti-VCAM-1 mAbs. In one [15 ] but not all experiments [20 ], CD8 T cells still managed to selectively infiltrate islets in both forms of treatment. A short-term, 4-week treatment of 10-week-old mice with anti-{alpha}4 subunit antibody resulted in significant and long-lasting suppression of disease [20 ]. Intriguingly, insulitis returned to a severe degree following suspension of anti-{alpha}4 mAb treatment, yet only 40% of the treated animals eventually become diabetic [21 ]. Finally, treatment of mice with mAbs against the integrin ß7 subunit and MAdCAM-1 led to significant, long-lasting protection against the spontaneous development of diabetes and insulitis [11 ].

A role for the leukocyte integrin {alpha}Lß2 (LFA-1) and its ligand intercellular adhesion molecule-1 (ICAM-1) is more controversial; in one study, anti-{alpha}Lß2 and anti-ICAM-1 mAbs blocked adoptive transfer of diabetes [22 ], whereas in another study, anti-ICAM-1 mAbs only minimally inhibited adoptive transfer of disease [15 ].

The use of exploring the therapeutic benefit of a single integrin or subset of integrins may be limited by the existence of compensatory cell-adhesion pathways. Here, we resolve this issue by simultaneously inhibiting the major cell-adhesion pathways involving {alpha}4, ß7, and ß2 integrins responsible for the infiltration of leukocytes into the inflamed pancreas. We evaluated the effectiveness of combinational treatment in inhibiting diabetes and determined the fate of leukocytes responsible for the insulitis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
NOD mice
The establishment of a colony of NOD/LtJ mice, obtained from Jackson Laboratories (Bar Habor, ME), has been described previously [4 ]. In this colony, approximately 70% of females develop diabetes between the ages 90 and 250 days. Diabetes was defined as heavy glycosuria for 3 consecutive days as detected by Testape (Eli Lilly, Indianapolis, IN) and confirmed by a hyperglycemic index of >12 mM in tail vein blood (Medisense blood glucose meter). Differences in diabetes incidence between treatment and control groups were analyzed by two-sample t-tests.

Antibodies
The anti-mouse mAbs anti-ß7 subunit (FIB504.84) [23 ], anti-{alpha}4 subunit (PS/2) [24 ], anti-ß2 subunit (2E6) [25 ], anti-VCAM-1 (M/K1.9) [24 ], anti-ICAM-1 (BE29G1) [26 ], and anti-{alpha}4ß7 complex-specific mAb DATK32 [27 ] used in therapy were prepared as ascites from hybridomas purchased from the American Type Culture Collection (Manassas, VA). The rat hybridoma cell line MECA-367 [27 ], which secretes a mAb against mouse MAdCAM-1, was kindly provided by Dr. Eugene Butcher (Stanford University, Stanford, CA). Normal rat immunoglobulin G (IgG) was purchased from Sigma (St. Louis, MO). Antibodies used for immunohistochemical staining included rat mAbs to mouse macrophages (M/170) [28 ], CD4 (H129.19) [29 ], and CD8 (53-6.72) [30 ], supplied as culture supernatants by Dr. H. Georgiou (Walter and Eliza Hall Institute, Melbourne, Australia). Guinea pig anti-insulin serum was prepared by Dr. J. Crossley (Department of Paediatrics, University of Auckland, New Zealand) and is specific for insulin [31 ].

Purified rabbit polyclonal antibodies (1 mg/ml) to mouse interleukin (IL)-4 and interferon-{gamma} (IFN-{gamma}) were obtained from Peprotech (Rocky Hill, NJ). A rat anti-IL-10 mAb (JES5-16E3) was obtained from PharMingen (San Diego, CA). Polyclonal antibodies to mouse macrophage inducible nitric oxide synthase (iNOS; rabbit IgG fraction, 1 mg/ml) [32 33 34 35 ] were prepared by Dr. C. Nathan (Cornell University Medical College, Ithaca, NY) [36 ] and were supplied by Upstate Biotechnology (Lake Placid, NY).

Antibody blockade of diabetes induced by adoptive transfer
Adoptive transfer of diabetic splenocytes was performed as demonstrated earlier [37 ]. Briefly, a single-cell suspension of splenocytes (98% viable), depleted of red cells, was prepared from four diabetic, female NOD mice. Splenocytes (2x108) were incubated at 37°C for 30 min with 100 µg PS/2 mAb alone or a combination of 100 µg each of the PS/2, DATK32, 2E6, FIB504.84, MECA 367, M/K1.9, and BE29G1 mAbs. Control splenocytes were incubated with 100 µg rat IgG control antibody. Antibody-treated splenocytes (2x107) were injected intravenously in 100 µl phosphate-buffered solution (PBS) into 8- to 10-week-old recipient male mice that had been sublethally irradiated (750 rads) the day before, and treatment groups contained six to seven mice. Following adoptive transfer (24 h later), animals received intraperitoneal (i.p.) injections of 100 µg PS/2 mAb, rat IgG control, or the above mAb combination, thrice per week until 52 days post-transfer or diabetes onset.

Antibody blockade of spontaneous diabetes
Groups of six to nine female NOD mice received i.p. injections of 100 µg PS/2 mAb, the above mAb combination, or rat IgG control, beginning at 7, 40, or 70 days of age. Antibodies were administered every other day for 4 weeks, and animals were monitored for spontaneous onset of diabetes or until day 250.

Immunohistochemical procedures
Serial frozen sections (6–8 µm) prepared from different levels of the pancreas were thaw-mounted on glass slides, fixed in cold acetone for 10 min, and stored at -20°C until use. For histochemical staining, sections were re-fixed in cold acetone and stained with haematoxylin and eosin (H&E) to monitor islet infiltration.

Serial sections were stained for CD4 and CD8 cells and macrophages, by the immunoperoxidase procedure, as demonstrated previously [4 ] with minor modifications. Following the blocking step, sections were incubated with mAbs to CD4, CD8, and macrophages overnight at room temperature in a moist chamber. After washing, sections were incubated with rabbit anti-rat IgG-biotin for 1 h at 37°C, followed by quenching with 0.3% hydrogen peroxide in methanol. Sections were developed with freshly prepared streptavidin-peroxidase (A+B, Vectastain) at RT for 30 min, followed by the addition of DAB (3-3', diaminobenzidine tetrahydrochloride) and H2O2 substrate for 3–5 min. Sections were counterstained with haematoxylin and viewed by light microscopy. As controls, primary antibodies were substituted with normal sera. For immunostaining of insulin, frozen and paraffin-embedded sections were developed using a two-step immunoperoxidase procedure involving staining with guinea pig anti-insulin serum and detection with peroxidase-conjugated, rabbit anti-guinea pig IgG. For controls, the primary sera were substituted with normal guinea pig sera or pre-adsorbed with insulin.

The immunohistochemical procedure for the analysis of cytokine expression has been described previously [4 , 33 ]. Sections were washed in excess PBS, pH 7.5, containing 0.3% saponin (Sigma), re-fixed in cold acetone, equilibrated in serum for 1 h at 37°C, and incubated with rabbit anti-IL-4 (1:50), rabbit anti-IFN-{gamma} (1:50), and rat anti-IL-10 (1:50) for 18 h at 4°C. They were then incubated with goat anti-rabbit and rabbit anti-rat IgG biotin, respectively (1:200, Jackson ImmunoResearch Laboratories, West Grove, PA), for 1 h at 37°C and then with streptavidin Texas Red (1:200, Jackson Laboratories) for 1 h at 37°C. Sections were washed, mounted with glycerol:PBS, and examined by fluorescence microscopy.

Immunohistochemical analysis of iNOS expression was carried out as described previously [34 , 35 ]. Sections were blocked with normal sheep serum, incubated with anti-iNOS (1:200) for 16 h at RT, and followed by incubation with goat anti-rabbit IgG biotin (1:200, Jackson Immunoresearch Laboratories) for 1 h at 37°C. Sections were finally incubated with streptavidin-Texas Red (1:200, Jackson Immunoresearch Laboratories) for 1 h at 37°C, washed, and mounted with glycerol:PBS before microscopic examination. For controls, primary antibodies were replaced with normal rabbit IgG at equivalent dilutions. For an additional control, the primary rabbit antiserum was absorbed with an excess of the homologous cytokine.

Appropriate fields of each section were photographed, and images were transferred to a photo compact disc and assembled in Adobe Photoshop 4.0 (Adobe Systems, San Jose, CA).

Histopathological evaluation of insulitis
For the adoptive-transfer experimental group, the severity of insulitis was determined at the onset of diabetes or 52 days post-transfer in those animals that did not become diabetic. In the case of the spontaneous diabetic group, the severity of insulitis was determined when mice were 250 days old or at onset of diabetes. The islets were graded on a scale of 0–4 for the degree of insulitis, as shown earlier [4 , 5 ]. Accordingly, no infiltration = grade 0; minimal focal infiltration = grade 1; peri-islet infiltration of <25% = grade 2; peri-islet infiltration and intra-islet infiltration of <50% = grade 3; and extensive intra-islet infiltration involving >50% of islet area = grade 4. At least 10 islets from different levels of the pancreas of each animal were scored. The insulitis score (%) for each group was calculated as follows: sum of (1xnumber of islets with grade 1; 2xnumber of islets with grade 2; 3xnumber of islets with grade 3; 4xnumber of islets with grade 4) divided by 4 x total number of islets scored. The ratio obtained was expressed as a percentage, and the mean (±SE) insulitis score (%) was recorded for the treatment and control group.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Combinational therapy with anti-CAM mAbs completely prevents the adoptive transfer of Type 1 diabetes
We first examined whether blockade of all major cell-adhesion pathways, by which leukocytes infiltrate the inflamed pancreas, has an enhanced therapeutic benefit in inhibiting adoptively transferred diabetes, compared with anti-integrin {alpha}4, ß2, and ß7 subunit monotherapies described previously [11 , 15 , 18 19 20 21 22 ]. Diabetic splenocytes from female NOD mice were treated in vitro with a blocking mAb against the integrin {alpha}4 subunit (PS/2) alone; with a combination of mAbs against {alpha}4ß7 (DATK32), the ß2 (2E6), ß7 (FIB504.84), and {alpha}4 (PS/2) subunits, MAdCAM-1 (MECA-367), VCAM-1 (M/K1.9), and ICAM-1 (BE29G1); or with normal rat IgG as a control. The splenocytes were adoptively transferred into irradiated male recipients, followed by in vivo treatment with the same antibodies. Administration of the combination of seven anti-CAM mAbs was of greater therapeutic benefit because 63% (5/8) of mice treated with the anti-{alpha}4 subunit mAb became hyperglycemic, whereas no mice in the combined antibody group became diabetic when followed until 52 days post-transfer (Table 1 ). In contrast, 89% (6/7) of the control rat IgG-treated group developed diabetes. There was a statistically significant difference (P=0.026) in the day of diabetes onset between the anti-{alpha}4 subunit mAb-treated mice that developed diabetes and control groups, where the average day of onset for overt diabetes was 43 (anti-{alpha}4 mAb) versus 31 (control), respectively, following post-transfer of diabetes-inducing splenocytes (Table 1) . Thus anti-{alpha}4 mAb monotherapy appears to delay disease onset.


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  		Table 1. The Incidence of Adoptively Transferred Diabetes in anti-CAM mAb-Treated NOD Micea

 
Combinational therapy with anti-CAM mAbs effectively inhibits spontaneous diabetes but only when administered from an early age
To examine whether combined, anti-CAM mAb treatment was as effective in preventing spontaneous diabetes in NOD mice as it was in inhibiting adoptively transferred disease, we administered antibodies to female NOD mice at different stages of disease progression. When short-term (4 wk) treatment was administered from 7 days of age, 33% (3/9) of mice treated with the anti-{alpha}4 subunit antibody and 17% (1/6) of those treated with the anti-CAM mAb combination became diabetic between 141 and 241 days of age (Table 2 ). In contrast, 71% (5/7) of animals injected with normal rat IgG became diabetic between 107 and 171 days of age. Hence, anti-CAM therapies can decrease disease incidence when administered at an early age. In contrast, antibody blockade was much less successful when used to treat advanced disease. Thus, when treatment was initiated on day 40, 50% (3/6) of the anti-{alpha}4 subunit mAb-treated and 60% (3/5) of the antibody combination-treated animals became diabetic compared with 83% (5/6) of control animals. Further, in those animals that developed disease, there was little difference in disease onset between anti-CAM, mAb-treated animals (92–248 days of age) and controls (98–250 days of age).


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  		Table 2. The Incidence of Spontaneous Diabetes in anti-CAM mAb-Treated NOD Micea

 
Anti-integrin {alpha}4 subunit mAb provides some protection even when administered just prior to diabetes onset
Antibody treatment administered from 70 days of age, only 3 weeks before normal diabetes onset, resulted in 50% (3/6) of animals from the anti-{alpha}4 subunit mAb group and 100% (6/6) of the antibody combination group, developing overt diabetes between 94 and 250 days of age (Table 2) . For comparison, 83% (5/6) of the control animals developed diabetes between 128 and 159 days of age. Thus, specifically blocking {alpha}4-mediated adhesion pathways has a beneficial effect even when administered at the end of the pre-diabetic period, whereas combinational antibody treatment was not protective.

Anti-CAM mAb treatment inhibits insulitis
Pancreases were examined histologically to determine whether antibody blockade inhibited the severity of insulitis as a mechanism to explain the decreased incidence of diabetes. In the adoptive-transfer model, those mice treated with the antibody combination had a reduced mean insulitis score (57%) at 52 days post-transfer compared with the rat IgG-treated control group (89%; Fig. 1A ). The insulitis was mostly restricted to the periphery of the islets with minimal intra-islet infiltration (Fig. 2a ), whereas peri- and intra-islet insulitis was observed in control rat, IgG-treated mice (Fig. 2c) . Anti-{alpha}4, subunit-treated animals had a mean insulitis score of 63% with peri- and intra-islet insulitis being detectable (Fig. 2b) . For statistical analysis, a mixed model was used, allowing different variances, but the only explanatory variable was group. There was evidence of a difference in the amount of insulitis in the three groups (F2,15=13.9, P=.0004), with the PS/2 and mAb combination treatment groups differing from the rat IgG control (both, P=0.001). In the analysis of insulitis in the spontaneous diabetes group, the data were only analyzed for treatment initiated at day 7, because delayed treatments had no observable beneficial effect on insulitis scores. When antibody treatment was initiated from day 7, the mean insulitis scores for the anti-{alpha}4 subunit mAb (35%; Fig. 2k )- and combination mAb (23%; Fig. 2j )-treated groups were similar and less than that of the control group (55%; Figs. 1B and 2l ). In contrast, insulitis was at least as severe as the control group when treatment was initiated from day 40, evidenced by mean insulitis scores of 72% (anti-{alpha}4 subunit mAb), 78% (combination mAbs), and 51% (normal rat IgG) in the various groups. The lower insulitis score for the control group may reflect the heterogeneity of pathogenesis at early stages of disease, causing insulitis scores to be slightly more erratic. The difference becomes negligible at late stages of disease, as evidenced by a high degree of insulitis when treatment was begun at 70 days of age in all groups—73% (anti-{alpha}4 subunit mAb), 77% (combination mAbs), and 76% (normal rat IgG; Fig. 1B ).



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  		Figure 1. Anti-CAM mAb treatment reduces mean insulitis scores but only when administered from an early stage of disease. Insulitis scores were obtained from a minimum of three mice. (A) Scores in the adoptive-transfer model were obtained at the onset of diabetes (refer to Table 1 ) or 52 days post-transfer in those animals that did not become diabetic. Mice were treated with PS/2 mAb (solid bar), mAb combination (hatched bar), and rat IgG control (open bar). (B) In the case of the spontaneous diabetic group, the severity of insulitis was determined when mice were 250 days old or at the onset of diabetes (refer to Table 2 ), following antibody treatment initiated at days 7, 40, and 70. Mice were treated with PS/2 mAb (solid bar), mAb combination (shaded bar), and rat IgG control (open bar).

 


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  		Figure 2. Anti-CAM mAb blockade inhibits leukocyte infiltration into the pancreas of NOD mice. For the adoptive-transfer model (a–i), frozen sections illustrated were prepared at 52 days post-transfer for the anti-CAM mAb combination-treated group or at the time of diabetes onset for the control group. In the case of the spontaneous diabetes model (j–r), sections illustrated were prepared at 250 days of age from mice that did not become diabetic following treatment with the anti-CAM mAb combination or at the time of diabetes onset for the control group. Sections prepared from animals treated with the antibody combination (a and j), anti-{alpha}4 subunit mAb (b and k), and control mice (c and l) were stained with haematoxylin and eosin. Sections from animals treated with the antibody combination were immunostained with mAbs against CD4 (d, g, m, and p) and CD8 cells (e, h, n, and q) and with macrophages (f, i, o, and r). No immunostaining was obtained when the primary antiserum was replaced by normal rat serum or when the secondary antibody was omitted. Original magnification, 40x.

 
Anti-CAM mAb treatment inhibits the entry of three major leukocyte subsets into islets
We next sought to determine whether combined, anti-CAM treatment prevented diabetes by blocking the entry of CD4 and CD8 T cells and macrophages into the islets or by selectively inhibiting the entry of a certain leukocyte subset(s), thereby causing immune deviation. Anti-CAM treatment led to almost complete exclusion of each of the CD4, CD8, and macrophage subsets from islets in adoptively transferred (Fig. 2d 2e 2f) and spontaneous (treatment initiated at day 7) forms of disease (Fig. 2m 2n 2o) . Inhibition of leukocyte entry into islets was generally more effective in the adoptive-transfer model. Leukocytes were almost exclusively restricted to the peri-islet region. In marked contrast, islets from the rat IgG-treated control groups (Fig. 2g 2h 2i and p-r ) were extensively infiltrated with all three major leukocyte subsets. In accord, ß-cell function was grossly impaired in control animals, as evidenced by a lack of insulin expression (Fig. 3A a ), whereas the levels of insulin in islets of combinational anti-CAM, mAb-treated animals appeared very similar to that of non-diseased animals (Fig. 3A , compare b and c).



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  		Figure 3. Analysis of islet pathology. (A) Insulin expression is retained in pancreatic islets of mice treated by combination mAb therapy. Pancreatic sections were obtained 52 days post-transfer for the anti-CAM mAb combination-treated group. Immunohistochemical staining for insulin granules (brown/yellow) revealed similar levels of insulin expression in non-diseased (c) and combined antibody (b)-treated mice, whereas insulin was almost undetectable in control diabetic mice (a). No immunostaining of insulin was obtained when the primary antibody was replaced with normal guinea pig serum or preadsorbed with excess insulin. (B) Peri-islet leukocytes in combined, antibody-treated mice are largely inactive. Sections prepared from diabetic control mice (a–d) and from combined, antibody-treated mice, 52 days following adoptive transfer (e–h), were stained with antibodies against IFN-{gamma} (a and e), IL-4 (b and f), IL-10 (c and g), and iNOS (d and h). Original magnification, 20x (for IFN-{gamma} and IL-4) and 40x (for IL-10 and iNOS).

 
Peri-islet leukocytes trapped by anti-CAM therapy are largely inactive
It seemed remarkable that leukocytes remained trapped within the peri-islet region following therapy, even 52 days following adoptive transfer. We decided to determine whether these leukocytes were still in a state of activation, as evidenced by their ability to secrete inflammatory cytokines and iNOS. A temporal relationship between immune-cell influx and the expression of iNOS, IL-4, and IFN-{gamma} has previously been demonstrated following adoptive transfer of diabetic spleen cells [4 ]. Levels of inflammatory cytokines and iNOS were examined in sections from each animal in each of the treatment groups and were found to be consistent.

IFN-{gamma}-, IL-4-, and iNOS-positive cells were readily detectable in the islets of control animals, whereas IL-10 was very weakly expressed (Fig. 3B 3a-d ). In marked contrast, only IL-10 expression was readily detectable in the islets of combined, antibody-treated mice (Fig. 3B 3e-h ).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The current study was undertaken to determine whether antibody blockade of the major cell-adhesion pathways used by leukocytes to access pancreatic islets would deliver enhanced therapeutic benefit. The rationale was based on the fact that the composition of the leukocyte infiltrate is complex with all major leukocyte types involved, including CD4 and CD8 T cells, B cells, macrophages, neutrophils, and dendritic cells [3 ], thereby implicating the use of multiple adhesion pathways. Blockade of the {alpha}4ß7/{alpha}4ß1/MAdCAM-1/VCAM-1 and ß2/ICAM-1 cell-adhesion pathways completely blocked adoptive transfer of diabetes, where treatment was continued for the duration of the experiment. Combined, short-term (4-week) antibody treatment almost completely abolished spontaneous diabetes (reduced incidence from 71% to 17%) when administered from 7 days of age. In contrast, monotherapy with the anti-{alpha}4 subunit mAb was less effective in treating adoptively transferred (incidence reduced from 89% to 63%) and early spontaneous disease (incidence reduced from 71% to 33%). We cannot exclude the possibility that higher doses of anti-{alpha}4 subunit mAb might be more effective, however an identical administration protocol using the PS/2 (anti-{alpha}4 subunit) mAb reduced the incidence of virus-facilitated, experimental, allergic encephalomyelitis by 90–100%, suggesting that the antibody must be close to saturating levels. In a less-rigorous treatment approach to the one described in the present study, a dosing regime of 75 µg PS/2 mAb every 3–4 days was found to maintain maximal coating of integrin {alpha}4+ cells in the peripheral blood, lymphoid organs, and bone marrow and protected against disease for the 22–26 days the antibody was administered [21 ]. Whether protection could be maintained in the long-term by continued administration of the antibody was not examined, and in fact, the disease returned sharply following suspension of antibody administration. In our treatment regime, which is the most prolonged to date, 100 µg PS/2 mAb was given thrice per week until 52 days post-transfer or diabetes onset. Despite the increased antibody dosage, 63% of mice became diabetic between days 37 and 49 post-transfer, however the disease was significantly delayed in those mice that succumbed to disease. Similar results have been demonstrated previously. Thus, Tsukamoto et al. [19 ] showed a 15% incidence of disease with 100 µg PS/2 mAb given three times a week when mice were followed for 42 days. We had a 25% incidence of disease at this time point. Other studies demonstrating complete protection by anti-{alpha}4 mAb treatment against adoptively transferred disease only followed mice for 35 days post-transfer, albeit all control mice were diseased at this time [20 ]. In accord, none of the anti-{alpha}4 mAb-treated mice in the present study had developed diabetes at 35 days post-transfer, whereas two-thirds of control mice had disease. Baron et al. [15 ] followed their anti-{alpha}4 mAb-treated mice for 38 days post-transfer with 10% developing diabetes, and in comparison, our incidence at this time was 12.5%. Fabien et al. [22 ] administered a large dose of 500 µg anti-{alpha}4 subunit mAb 3 times a week, 1 week before transfer and 2 weeks after. Despite this large dose of antibody, 40% of mice had become diabetic by day 45. Our results, together with the above studies, indicate that anti-{alpha}4 mAb monotherapy is unlikely to prevent adoptively transferred disease in the long-term in a large proportion of mice, as is the case for spontaneous disease.

Despite the differences in therapeutic efficacy, there was little difference in the insulitis scores between animals treated with the anti-{alpha}4 subunit mAb versus the antibody combination, albeit there was always slightly greater intra-islet infiltration with anti-{alpha}4 mAb monotherapy. This, together with the strong inhibition of early, spontaneous diabetes, suggests that {alpha}4 integrins function as the major receptor in the infiltration process.

We find that our results in treating spontaneous disease agree with previous published studies, demonstrating that in vivo blockade of {alpha}4 integrins can inhibit the natural progression of diabetes in NOD mice. We chose a different schedule of antibody administration, compared with that demonstrated by Yang et al. [20 ], to treat spontaneous disease. They treated newborn mice, whereas we treated 1-week-old mice for a 4-week period. In accord, they found that a 4-week treatment with anti-{alpha}4 mAb was less effective (30% incidence vs. 84% for control) when delayed until 10 weeks of age. Our results are similar to those of Tsukamoto et al. [19 ], who demonstrated that a 3-week treatment with anti-{alpha}4 and anti-VCAM-1 mAbs from 2 weeks of age only lowered the incidence of disease from 58% to 38%. In contrast, they showed that continuous treatment with anti-{alpha}4/VCAM-1 antibodies from 2 or 10 weeks until 30 weeks of age (200 µg each mAb, thrice per week) provided complete or significant (90%) protection. In summary, anti-{alpha}4 mAb therapy may be optimally effective as a short-term treatment when administered from birth but loses its therapeutic potential when treatment is delayed by even 1 week unless given continuously.

We have demonstrated here that combinational treatment targeting ß2 integrin pathways in addition to {alpha}4 pathways is of therapeutic benefit when treatment is initiated from 7 days of age. A contribution from blocking ß2-integrin pathways is demonstrated in previous studies where treatment with anti-{alpha}L subunit mAb, alone or in combination with anti-ICAM-1 mAb, is effective at decreasing the incidence of diabetes and insulitis in the adoptive transfer and spontaneous models [22 , 38 , 39 ]. It is important that anti-{alpha}4 mAb and combined mAb treatment lead to long-lasting suppression of disease in a majority of animals when administered from an early age.

It is surprising that anti-{alpha}4 mAb treatment appeared to exert therapeutic potential even when administered late in the pre-diabetic period, just prior to diabetes onset, when insulitis is already well established. Disease incidence was reduced from 83% to 50%, whereas the antibody combination had no effect. Uniyal et al. [40 ] have proposed that {alpha}4 and {alpha}5 integrins play a role in intra-islet infiltration. It is possible that the antibody combination may upset this balance, making infiltrated leukocytes more dependent on {alpha}5 integrins and thereby potentially preventing their removal.

This study is the first to analyze the long-term fate of CD4 and CD8 and macrophage cell types following anti-CAM therapy. The degree of intra-islet infiltration, rather than the overall degree of insultis, correlated well with the incidence of diabetes. Thus, anti-{alpha}4 therapy to treat the early stages of disease progression resulted in a higher incidence of diabetes, which correlated with greater intra-islet infiltration than that observed using combined antibody treatment (unpublished results). Leukocytes remaining at islets after combined mAb therapy were found to reside predominantly within a peri-islet location. Remarkably, the major CD4 and CD8 T-cell and macrophage subsets assumed this peri-islet location, both 52 days following adoptive transfer and 215 days after suspension of antibody treatment in the spontaneous model of diabetes. This contrasts with a short, two-week blockade with anti-{alpha}4 mAb, where intra-islet infiltration in adoptively transferred diabetes progressed to a severe degree once mAb treatment was suspended [21 ]. Thus, treatment may need to be sustained for at least 4 wk to prevent pathogenic leukocytes from taking up an intra-islet location.

Why don’t peri-islet leukocytes migrate into the islets following suspension of combined anti-CAM therapy? The answer may be in the discovery that peri-islet cells were largely inactive, because they had stopped expressing Th1 (IFN-{gamma}) and Th2 (IL-4) cytokines or iNOS. Reddy et al. [4 ] have revealed that intra-islet CD4 cells in the adoptive-transfer model produce IL-4, whereas peri- and intra-islet macrophages produce IFN-{gamma} and iNOS. We propose that peri-islet leukocytes have become trapped within this location. They are unable to enter the islet to be stimulated with islet autoantigens, hence remain as inactive exiles, perhaps forming an impenetrable barrier preventing the entry of active, autoantigen-reactive cells. We cannot exclude the possibility that there is a dynamic exchange between leukocytes from the periphery and those in a peri-islet position. The only examined cytokine to be produced by peri-islet leukocytes was IL-10, which may have a therapeutic effect as a result of its immunosuppressive properties [41 ]. IL-10 has been shown to have a beneficial effect in animal models of rheumatoid arthritis [42 ], inflammatory bowel disease [43 ], and experimental autoimmune encephalomyelitis [44 ] by generating regulatory/suppressor cells or by suppressing T-cell effector functions. In contrast, the effects of IL-10 in the immunoregulation of Type I diabetes appear to be paradoxical [45 ]. IL-10 appears essential for an early phase of diabetes in the NOD mouse but protects against development of disease at later stages [46 ]. Over-expression of IL-10 in pancreatic islet {alpha} or ß cells accelerates the onset of diabetes [47 , 48 ]. Conversely, systemic administration or regulated T-cell production of IL-10 protects against disease [49 50 51 52 ]. This latter finding is of particular interest given that IL-10 was expressed by the by peri-islet leukocytes of treated mice in the present study.

In conclusion, we have shown that short-term, combinational, anti-CAM antibody therapy is effective in providing long-lasting suppression of diabetes but only if administered from an early age. Once insulitis is well-established, anti-{alpha}4 subunit monotherapy appears to be more effective. Disease suppression may involve the entrapment of major leukocyte subsets rendered inactive within a peri-islet location, forming a barrier to the entry of active, disease-forming cells.


   ACKNOWLEDGEMENTS
 
This work was supported in part by grants from the Lottery Grants Board of New Zealand, the Royal Society of New Zealand, the Health Research Council of New Zealand, the Wellcome Trust, and the Marsden Fund. G. W. K. was supported by a James Cook Research Fellowship funded by the Royal Society of New Zealand. We are grateful to Joanna Stewart (Department of Community Health) for assistance with the statistical analysis.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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