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The Journal of Immunology, 2001, 167: 6637-6643.
Copyright © 2001 by The American Association of Immunologists

IFN-{gamma} Affects Homing of Diabetogenic T Cells1

Alexei Y. Savinov*, F. Susan Wong{dagger} and Alexander V. Chervonsky2,*

* The Jackson Laboratory, Bar Harbor, ME 04609; and {dagger} Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
IFN-{gamma} is a cytokine with pleiotropic functions that participates in immune and autoimmune responses. The lack of IFN-{gamma} is known to delay the development of autoimmune diabetes in nonobese diabetic (NOD) mice. Splenocytes from diabetic NOD and IFN-{gamma} knockout (KO) NOD mice transfer diabetes into NOD recipients equally well. However, adoptive transfer of diabetogenic T cells from NOD mice into NOD.IFN-{gamma}-KO or NOD mice lacking {beta}-chain of IFN-{gamma} receptor (NOD.IFN-{gamma}R{beta}-KO) appeared to be much less efficient. We found that IFN-{gamma} influences the ability of diabetogenic cells to penetrate pancreatic islets. Tracing in vivo of insulin-specific CD8+ T cells has shown that homing of these cells to the islets of Langerhans was affected by the lack of IFN-{gamma}. While adhesion of insulin-specific CD8+ cells to microvasculature was normal, the diapedesis was significantly impaired. This effect was reversible by treatment of the animals with rIFN-{gamma}. Thus, IFN-{gamma} may, among other effects, influence immune and autoimmune responses by supporting the homing of activated T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Insulin-dependent diabetes mellitus (IDDM)3 develops spontaneously in genetically prone humans and experimental animals. Multiple autoimmune effector mechanisms lead to destruction of the insulin-producing {beta} cells by triggering several apoptotic pathways (1). In addition to tumor necrosis family factors (Fas ligand and TNF-{alpha}) (2, 3, 4, 5) that can directly trigger apoptosis in {beta} cells, the proinflammatory cytokine IFN-{gamma} has also been associated with the development of IDDM. IFN-{gamma} has been detected in the islets of nonobese diabetic (NOD) mice in increasing amounts during progression of diabetes (6). Transgenic expression of IFN-{gamma} driven by the insulin promoter in {beta} cells caused progressive lymphocyte-mediated destruction of pancreatic islets in diabetes-resistant BALB/c mice (7, 8). Expression of IFN-{gamma} was not directly toxic to {beta} cells, but rather affected diabetogenesis by promoting the recruitment and activation of cytotoxic T cells and macrophages. Inactivation of IFN-{gamma} by specific Abs (9, 10) or by IFN-{gamma} receptor-Ig fusion molecules (11, 12) significantly reduced the incidence of diabetes. Targeted mutation of the IFN-{gamma} gene (13) and transfer of the mutation onto the NOD genetic background (14) revealed that IFN-{gamma} deficiency did not prevent development of diabetes, but delayed the onset of the disease. This was a clear indication that multiple effector mechanisms are involved in diabetes development and that other pathogenic mechanisms may compensate for the absence of IFN-{gamma}. Interestingly, inactivation of the IFN-{gamma} receptor {alpha}-chain (15), but not {beta}-chain (16), led to complete prevention of diabetes, possibly because a gene adjacent to the IFN-{gamma} receptor {alpha}-chain gene inherited from 129 strain of mice provided the resistance (17).

Although the function of IFN-{gamma} in diabetogenesis can be compensated by other mechanisms, those steps in development of the disease that are critical for its progression and are under the control of this cytokine have yet to be revealed. IFN-{gamma} is a cytokine that has pleiotropic action and influences the transcription of hundreds of genes (18). Numerous immune defects were found in IFN-{gamma}-negative mice (13, 19). In IDDM, IFN-{gamma} may potentially influence the development of autoimmune Th1 CD4+ T cells (20, 21), affect the sensitivity of {beta} cells to destruction (e.g., by up-regulation in concert with IL-1 of expression of the proapoptotic receptor Fas) (4), and influence autoantigen presentation by both MHC class I and class II molecules (22, 23, 24, 25).

The efficiency of the transfer of diabetes by splenocytes from diabetic IFN-{gamma} knockout NOD (NOD.IFN-{gamma}-KO) mice into NOD recipients did not differ from the transfer of diabetes by splenocytes from diabetic IFN-{gamma}-sufficient NOD donors (14). However, we found that reciprocal transfer of diabetogenic splenocytes into NOD.IFN-{gamma}-KO recipients induced diabetes in a small number of animals. In this study, we show that IFN-{gamma} plays an important role in the control of the homing of diabetogenic T cells to the islets.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Mice

NOD/LtJ (NOD), NOD.CB17-Prkdcscid/J (NOD-scid), NOD.129S7(B6)-IFN-{gamma}tm1Ts (NOD.IFN-{gamma}-KO), NOD.129S7(B6)-IFN-{gamma}tm1Ts-Prkdcscid (NOD.IFN-{gamma}-KO-scid), BALB/cJ, and C.129S7-IFN-{gamma}tm1Ts mice were obtained from The Jackson Laboratory (Bar Harbor, ME). NOD.129-IFN-{gamma}R2tm1Cmb (NOD.IFN-{gamma}R{beta}-KO) mice were the generous gift from Dr. D. Serreze (The Jackson Laboratory). All congenic animals were back-crossed to NOD to at least N8 generation. All animals were housed in a specific pathogen-free research facility. Diabetes incidence was measured by monitoring glucose levels in urine using Diastix reagent strips (Bayer, Elkhart, IN). For genotyping of IFN-{gamma}-negative mice, genomic DNA from tails, pancreata, or spleens were used for PCR amplification with primers specific for wild-type or IFN-{gamma}-KO alleles (13). A 220-bp product from the wild-type IFN-{gamma} allele was amplified using a 5'-AGAAGTAAGTGGAAGGGCCCAGAAG-3' forward and 5'-AGGGAAACTGGGAGAGG AGAAATAT-3' reverse primers pair. For the amplification of a 375-bp product corresponding to the targeted IFN-{gamma} allele, 5'-TCAGCGCAGGGGCGCCCGGTTCTTT-3' forward and 5'-ATCGACAAGACCGGCTTCCATCCGA-3' reverse primers were used.

Adoptive transfer

Adoptive transfer of diabetes was performed by i.v. injection of 1.5 x 107 of splenocytes from diabetic animals into irradiated (725 rad, 24 h in advance) recipients. Both males and females 4–11 wk of age were used. No sex- or age-dependent difference was found in the incidence or time of onset of diabetes, and therefore results were pooled. Transfer into the host animals carrying the scid mutation was performed without irradiation. The recipients of adoptive transfers were monitored for 50 days for diabetes development.

Statistical analysis

Analysis of statistical significance of the observed differences between various groups of mice in performed experiments was done using SuperANOVA software (Microsoft, Seattle, WA), utilizing Fisher’s protected least significant difference (LSD) test at the significance level of 0.05. In addition, in adoptive transfer experiments, statistical significance of differences in the distributions of day at onset for two group pairs indicated in Table IGo was assessed by permutation tests using the likelihood ratio test statistic from the Cox proportional hazards model (26). With use of a permutation test, the analysis does not rely on the detailed assumptions of the Cox model.


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  		Table I. Development of diabetes after adoptive transfer of diabetogenic splenocytes is delayed in mice that lack IFN-{gamma} or IFN-{gamma}R{beta}1

 
Analysis of short-term survival and proliferation of transferred cells

Lymphocytes were stained with the fluorescent dye CFSE (Molecular Probes, Eugene, OR), as described (27). A total of 107 labeled cells was injected i.v. into irradiated (725 rad, 24 h in advance) animals. Eight days after transfer, animals were sacrificed, and splenocytes and lymph node cells were isolated, stained with Red613-conjugated anti-CD4 Ab (Life Technologies, Rockville, MD) and PE-conjugated anti-CD8 Ab (BD PharMingen, San Diego, CA), and analyzed on FACSCalibur flow cytometer using the CellQuest software (both BD Biosciences, Mountain View, CA).

Insulin-specific CD8+ cells

Insulin-specific (IS), Kd-restricted T cells (IS-CD8+ cells) of the clone TGNFC8 (28, 29) were maintained in vitro in Click’s medium supplemented with 5% FCS (Sigma-Aldrich, St. Louis, MO) and 5 U/ml mouse IL-2. For antigenic stimulation, cells were exposed every 3 wk to irradiated (2000 rad) NOD-derived pancreatic islets or NOD splenocytes loaded with 0.01 mg/ml synthetic peptide derived from mouse insulin B chain, amino acids 15–23 (LYLVCGERG) (28), produced by Research Genetics (Huntsville, AL). Pancreatic islets were isolated by collagenase inflation method, as described (30), and handpicked in HBSS (Life Technologies) after purification on Histopaque 1119 (Sigma-Aldrich) gradient (31).

Cytotoxicity assay

Pancreatic islets isolated from NOD and NOD.IFN-{gamma}-KO mice were dispersed into single cell suspension by incubation in cell dissociation buffer (Life Technologies) for 1 h at room temperature, labeled with 100 µCi of Na251CrO4 (ICN Pharmaceuticals, Costa Mesa, CA) in 200 µl of Click’s medium containing 5% FCS for 2 h at 37°C, washed three times, and cocultured for 12 h in 96-well plates (104 targets/well in 200 µl of Click’s medium with 5% FCS) with effector IS-CD8+ cells at different E:T ratios. Cytotoxicity was measured by 51Cr release in 100-µl aliquots of cell-free supernatant using a gamma counter (Wallac, Turku, Finland) and calculated using the following formula: percentage of specific cytotoxicity = (experimental release - spontaneous release)/(maximum release - spontaneous release) x 100%.

Morphometric analysis of IS-CD8+ cells homing to the pancreas

For trafficking studies, IS-CD8+ cells were harvested and counted, and 107 cells/ml were incubated for 30 min at 37°C in the dark in complete medium containing 5% FCS and 0.0075 mg/ml fluorescent dye didodecyl-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes). Stained cells were washed three times with PBS and injected i.v. at 107 cells per mouse into irradiated (725 rad, 24 h in advance) recipients. Twenty-four hours after injection of IS-CD8+ cells, animals were sacrificed and their spleens and pancreata were removed, fixed in 0.1 M periodate-lysine-paraformaldehyde phosphate buffer, sucrose-saturated as described (32), and freeze molded in the OCT compound (Sakura Finetek, Torrance, CA). Cryostat sections (7 µm thick) of the entire pancreata were obtained in 60-µm intervals using a Leica CM1900 cryotom (Leica, Heerbrugg, Switzerland). Distribution of labeled CD8+ cells within the islets was examined using a fluorescent microscope DMLB (Leica). Each section was examined for the presence of labeled IS-CD8+ cells that were classified by their location (either at the islet entrance (attached to the capillary wall, or located in the isthmus) or inside the islet) and counted. The number of pancreatic islets examined was also recorded. Difference in mean numbers of labeled cells detected in each pancreatic location between groups of NOD and NOD.IFN-{gamma}-KO recipients was analyzed by Fisher’s protected LSD test at the significance level of 0.05.

Treatment with rIFN-{gamma}

In some experiments, animals were treated with mouse rIFN-{gamma} (PBL Biomedical Laboratories, New Brunswick, NJ) at 104 U/day i.p. in 0.1% BSA (Sigma-Aldrich) in PBS for 2 days before injection of IS-CD8+ cells. Control mice received 0.1% BSA solution.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Lack of IFN-{gamma} production by the host reduces the frequency and delays onset of adoptively transferred diabetes

IFN-{gamma}-deficient NOD mice develop spontaneous diabetes with a delay, compared with wild-type NOD mice (14). At the same time, splenocytes from diabetic NOD.IFN-{gamma}-KO mice transfer the disease into NOD recipients without any reduction of the transfer efficiency compared with splenocytes from IFN-{gamma}-sufficient NOD mice (14) (Table IGo). This result suggests that IFN-{gamma} is not required for the function of the mature diabetogenic effector T cells. Surprisingly, the reverse experiment in which diabetogenic NOD T cells are introduced into NOD.IFN-{gamma}-KO mice has not been previously reported. Thus, we performed a series of adoptive transfers of diabetogenic splenocytes using IFN-{gamma}-deficient mice as recipients (Table IGo, groups 2 and 4). We found that the transfer of splenocytes from diabetic NOD mice into IFN-{gamma}-deficient hosts was inefficient, because only 33% of recipients became diabetic within the observation period (up to 50 days). However, it could be argued that the more rapid onset of diabetes in IFN-{gamma}-sufficient compared with IFN-{gamma}-deficient recipients occurred because of the damage to the islets done by the ongoing inflammatory process in recipient NOD mice before the transfer. By the age of 7 wk, NOD mice develop significant insulitis, while NOD.IFN-{gamma}-KO mice are almost insulitis free (14). Thus, the difference in the mass of intact {beta} cells in the IFN-{gamma}-positive and IFN-{gamma}-negative recipients at the time of adoptive transfer could have influenced the difference in the onset of diabetes. To rule out that possibility, NOD mice homozygous for the scid mutation and either IFN-{gamma} sufficient (NOD-scid) or IFN-{gamma} deficient (NOD.IFN-{gamma}-KO-scid) were used as recipients. NOD-scid animals lack T and B cells, and develop neither insulitis nor diabetes (33). Upon adoptive transfer of diabetogenic splenocytes from NOD donors, 100% of IFN-{gamma}-sufficient NOD-scid mice developed diabetes by 5 wk after transfer, while only 23% of NOD.IFN-{gamma}-KO-scid hosts were diabetic 7 wk after transfer (Table IGo, groups 6–9). The patterns of diabetes development displayed by NOD and NOD.IFN-{gamma}-KO, and by IFN-{gamma}-positive and IFN-{gamma}-negative NOD-scid recipients after adoptive transfer were very similar. Thus, pre-existing damage does not appear to influence diabetes development upon adoptive transfer of diabetogenic T cells.

Several mechanisms could explain the inefficient transfer of diabetes into IFN-{gamma}-negative recipients. First, the absence of IFN-{gamma} can directly affect the population of diabetogenic effectors, decreasing their survival; second, IFN-{gamma}-deficient recipients could have altered presentation of certain islet-specific peptides; and third, IFN-{gamma} could be involved in regulation of the homing of diabetogenic effectors to the islets of Langerhans.

Host cells, but not donor cells, are affected by IFN-{gamma} deficiency

Do the transferred cells require host IFN-{gamma} to survive and/or exert their function, or are host tissues the primary targets influenced by the absence of IFN-{gamma}? To address these questions we performed the following experiments.

First, we needed to show that survival and proliferation of T cells are not affected in the IFN-{gamma}-negative recipients. To determine whether survival and/or homeostatic proliferation of peripheral T cells can be reduced in the absence of recipient-produced IFN-{gamma}, we transferred 107 lymph node cells from IFN-{gamma}-positive NOD mice into irradiated IFN-{gamma}+ or IFN-{gamma}- sex-matched mice. The donor cells were labeled before transfer with the cytoplasmic fluorescent dye CFSE. This allowed us to assess both survival and homeostatic proliferation of the transferred cells, since every division of CFSE-labeled cells leads to a 2-fold decrease in fluorescence in daughter cells (27). In three independent experiments, FACS analysis 8 days after transfer revealed no significant differences in CFSE+ populations recovered from spleens (Fig. 1Go), or from lymph nodes (data not shown) of IFN-{gamma}+ and IFN-{gamma}- hosts. In a representative experiment involving three NOD and three NOD.IFN-{gamma}-KO recipients, the absolute numbers (±SE) of surviving splenic CFSE+CD8+ cells were 3.1 x 104 ± 0.3 x 104 and 9.3 x 104 ± 3 x 104 per mouse, respectively. Clearly, short-term survival of donor cells was not diminished in NOD.IFN-{gamma}-KO recipients. Moreover, CFSE+ cells from both hosts displayed a similar distribution of CFSE fluorescence (Fig. 1Go), indicating that homeostatic proliferation of transferred T cells is also independent of IFN-{gamma}.



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  		FIGURE 1. IFN-{gamma} deficiency in NOD mice does not affect survival or homeostatic proliferation of the transferred T cells. FACS profiles of splenocytes (top panels) and CFSE-positive CD8+ cells (bottom panels) recovered from NOD and NOD.IFN-{gamma}-KO recipients of NOD lymph node cells 8 days after transfer.

 
Long-term survival of diabetogenic cells from NOD donors transferred into the IFN-{gamma}-deficient hosts was assessed by PCR-based detection of the presence of the wild-type IFN-{gamma} genomic DNA in the total DNA isolated from the spleens of IFN-{gamma}-negative recipients at different times after transfer. PCR amplification of donor-specific wild-type IFN-{gamma} allele showed the presence of donor cells in the spleens of NOD.IFN-{gamma}-KO hosts up to the time of the development of diabetes (Fig. 2Go). Thus, the long-term survival of transferred diabetogenic cells was also independent of IFN-{gamma}. Hence, the delay of the diabetes onset in IFN-{gamma}-deficient recipients cannot be explained by the poor survival of the diabetogenic effectors in the absence of IFN-{gamma} in vivo.



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  		FIGURE 2. At the time of the delayed diabetes onset in NOD.IFN-{gamma}-KO recipients, the presence of the donor NOD cells can be detected in the spleens of a recipient mice. Wild-type (donor) DNA was detected by PCR amplification of genomic DNA fragment absent from the NOD.IFN-{gamma}-KO recipient. Representative DNA amplification results are shown for three recipient mice sacrificed at indicated time points. *, Nondiabetic mouse; **, diabetic mice.

 
Second, to show that donor-derived effector cells do not need IFN-{gamma} to support their function, we used splenocytes from diabetic IFN-{gamma} receptor {beta}-chain-deficient NOD mice (NOD.IFN-{gamma}R{beta}-KO) to transfer the disease. In this case, donor cells are refractory to IFN-{gamma} signaling (34). NOD recipient mice acquired diabetes without delay, but at the same time NOD.IFN-{gamma}-KO mice demonstrated a delayed onset as well as a low efficiency (50%) of transfer (Table IGo, groups 10 and 11).

These results suggested that some cell types in the recipients rather than donor cells require IFN-{gamma} to allow efficient transfer of diabetes. That was further confirmed by injection of diabetogenic splenocytes into NOD.IFN-{gamma}R{beta}-KO mice that lack IFN-{gamma} signaling. The development of diabetes was delayed compared with normal NOD recipients (Table IGo, groups 1 and 5), indicating that recipient cells are affected by the lack of IFN-{gamma} signaling. These observations are in line with the results of Kanagawa et al. (17), who observed that NOD mice deficient for the IFN-{gamma} receptor {alpha}-chain showed significant delay in diabetes development upon adoptive transfer. Importantly, their recipients were back-crossed to NOD enough times to lose a gene responsible for resistance to spontaneous diabetes inherited from 129 strain. Yet the adoptive transfer of diabetes was delayed in these mice.

In our experiments, the delay in diabetes development upon adoptive transfer was less pronounced in NOD.IFN-{gamma}R{beta}-KO recipients compared with NOD.IFN-{gamma}-KO mice (Table IGo, groups 2 and 5). This can be explained by some additional genetic differences conferred with the targeted genes from 129 mice. Nevertheless, both KO mice show similar phenotype, and taken together all these observations suggest that the transferred cells do not require host-produced IFN-{gamma} to exert their function, and that the absence of IFN-{gamma} or its receptor expression by the recipient’s tissues provides a substantial level of resistance to the diabetogenic T cells. The results of adoptive transfer into NOD.IFN-{gamma}-KO-scid recipients (delayed onset of diabetes compared with NOD-scid; Table IGo, groups 6 and 7) suggest that nonlymphoid cells are critical targets for IFN-{gamma}. These nonlymphoid tissues may participate in Ag presentation and in homing of effector cells to the pancreatic islets.

IFN-{gamma} deficiency affects Ag presentation by {beta} cells

IFN-{gamma} regulates multiple steps in Ag processing (24, 35, 36) and presentation (22, 23, 37) by both MHC class I and MHC class II. It has been previously shown that exogenous IFN-{gamma} up-regulates MHC class I expression by the pancreatic {beta} cells (22, 23). Poor presentation of islet-specific Ags in the absence of IFN-{gamma} could contribute to the delay in the onset of diabetes. To address this issue, we used IS-CD8+ cells that recognize specific peptide derived from B chain of insulin (B15–23) (28). Pancreatic islet cells isolated from both NOD and NOD.IFN-{gamma}-KO mice were sensitive to IS-CD8+-induced cytotoxicity; however, islets from IFN-{gamma}-positive animals were killed more efficiently than islet cells from IFN-{gamma}-negative mice (Fig. 3Go). Difference in sensitivity to IS-CD8+-induced cytotoxicity of NOD vs NOD.IFN-{gamma}-KO-derived islets was significant (by Fisher’s protected LSD at significance level of 0.05) for all E:T ratios in each of the experiments shown in Fig. 3Go. Since the block of IFN-{gamma} signaling was reported to have little effect on the basal levels of MHC class I expression by {beta} cells in mice (23), it is reasonable to suggest that the presentation of a specific MHC class I-restricted insulin-derived peptide was affected by the absence of IFN-{gamma}. It remains to be seen whether presentation of different pancreas-specific peptides is affected by IFN-{gamma} deficiency; however, lack of IFN-{gamma} appears to affect Ag presentation by {beta} cells and thus contribute to the delay of diabetes development.



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  		FIGURE 3. Pancreatic islets from NOD.IFN-{gamma}-KO mice are less sensitive to IS-CD8+ cell-induced cytotoxicity than are the islets from NOD mice. 51Cr-Labeled pancreatic islet cells from NOD and NOD.IFN-{gamma}-KO mice were exposed to cytotoxic IS-CD8+ cells for 12 h. Specific cytotoxicity is shown for islet cells isolated from NOD (•) and NOD.IFN-{gamma}-KO ({circ}) mice. Results of two independent experiments are shown.

 
Altered homing of diabetogenic effector cells in IFN-{gamma}-deficient mice

In the experiments addressing long-term survival of diabetogenic cells in IFN-{gamma}-negative hosts, donor cells were detected in the spleens, but not in the pancreata, of diabetic recipients (Fig. 2Go). This observation suggested that donor diabetogenic cells do not efficiently populate the pancreas in a recipient that lacks IFN-{gamma}. To understand the effects that IFN-{gamma} might have on the migration of diabetogenic effector cells, we have chosen to use cloned Kd-restricted, mouse insulin B chain (amino acids 15–23)-specific CD8+ T cells isolated from a prediabetic NOD mouse (28, 29). These IS-C8+ cells provide a robust system for the studies of T cell homing, as they are highly diabetogenic, homogenous, and easily traceable into the islets (29). Injection of IS-C8+ cells into recipients of different genotypes revealed that their ability to cause diabetes was impaired in NOD.IFN-{gamma}-KO and NOD.IFN{gamma}R{beta}-KO recipients compared with NOD (Fig. 4Go). That was similar to what we have observed in transfer of diabetogenic splenocytes experiments (Table IGo). To further investigate the ability of IS-CD8+ cells to penetrate the islets, we performed a morphometric analysis of the islets from normal and mutant mice injected with IS-CD8+ cells. Distribution of DiI-labeled IS-CD8+ cells was compared in IFN-{gamma}-sufficient and IFN-{gamma}-deficient recipients 24 h after injection (Fig. 5Go). The distribution of the IS-CD8+ cells within the pancreata of NOD and NOD.IFN-{gamma}-KO mice was strikingly different: in IFN-{gamma}-sufficient mice, the majority of labeled cells was found inside the islets, while in IFN-{gamma}-deficient animals most of the labeled cells accumulated at the islet entrance (attached to the blood vessel walls or in the islet vascular isthmus) (Fig. 5GoA). The results of the analysis are shown in Table IIGo and Fig. 5GoB. The ratio of "cells inside the islets" to "cells at the entrance" was 3.1 ± 1.1 and 0.5 ± 0.1 in NOD and NOD.IFN-{gamma}-KO recipients, respectively. Clearly, IS-CD8+ cells were unable to penetrate the islets in the IFN-{gamma}-deficient hosts, although their attachment to vascular endothelium was not affected. Next we asked whether injection of rIFN-{gamma} would affect the pattern of IS-CD8+ cell distribution within the islets in NOD.IFN-{gamma}-KO recipients. Systemic application of IFN-{gamma} led to a partial increase in the proportion of cells that were able to penetrate the islets (the ratio of "inside" to "entrance" increased to 1.1 ± 0.1). Injection of a control solution containing 0.1% BSA did not affect the infiltration of the islets (Table IIGo).



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  		FIGURE 4. IS-CD8+ cells display reduced diabetogenicity when injected into recipients lacking IFN-{gamma} signaling. Ordinate, the proportion (%) of NOD, NOD.IFN-{gamma}-KO, and NOD.IFN{gamma}R{beta}-KO animals that developed diabetes after injection of 107 IS-CD8+ cells. Mean day of diabetes onset (±SE) is indicated. n, Number of recipients per group.

 


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  		FIGURE 5. Different distribution of labeled IS-CD8+ cells in IFN-{gamma}-sufficient and IFN-{gamma}-deficient NOD recipient mice. A, Comparison of pancreatic cryosections from NOD and NOD.IFN-{gamma}-KO recipients of the 107 DiI-labeled IS-CD8+ cells 24 h after transfer. Islets are marked by a dashed line. Cells were counted within areas relevant to a given islet (dotted rectangles). B, Quantification of the distribution of labeled IS-CD8+ cells within the islets of IFN-{gamma}-sufficient (open bars) and IFN-{gamma}-deficient (hatched bars) NOD mice. Pancreata of three mice of each genotype were analyzed. Data represent mean number ± SE of IS-CD8+ cells per islet at the indicated locations. See also Table IIGo.

 

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  		Table II. Adoptively transferred IS-CD8+ cells inefficiently penetrate the islets of IFN-{gamma}-deficient NOD recipients1

 
To cause diabetes, the transferred effector IS-CD8+ cells must directly contact their targets: pancreatic {beta} cells. To achieve this, IS-CD8+ cells must first migrate from the bloodstream into the islets. This process is complex and involves several steps of interaction between lymphocytes and endothelial cells of microcapillaries that include initial tethering and rolling, followed by activation-induced arrest and, finally, by extravasation or diapedesis (38). Initial tethering and rolling are mediated by the interaction of selectins and their ligands and the interaction of integrins with corresponding cellular adhesion molecules on the surfaces of lymphocytes and endothelial cells. Activation of integrins through the G protein-coupled chemokine receptors leads to increased adhesion and complete arrest of rolling. This arrest is followed by diapedesis, a step least understood in terms of its molecular mechanisms (39, 40). While homing of lymphocytes to the pancreatic islets is a critical step in the pathogenesis of diabetes, little is known about the regulation of the pancreas-specific homing of effector T cells, especially of CD8+ T cells. Morphometric analysis of islets from IFN-{gamma}-positive and -negative mice injected with IS-CD8+ cells (Table IIGo, Fig. 5GoB) demonstrated that initial homing events (adhesion to the blood vessels) were not affected by the lack of IFN-{gamma}, as the numbers of labeled cells found in both types of recipients did not differ significantly. However, extravasation of IS-CD8+ from capillaries into the islets was reduced dramatically. Thus, IFN-{gamma} produced by host cells is not required for tethering, rolling, and activation-dependent arrest of IS-CD8+ diabetogenic effectors. However, diapedesis appeared to be regulated by the IFN-{gamma}-dependent mechanism.

Since we have already established that the ability of the host to produce IFN-{gamma}, and not donor sensitivity to IFN-{gamma}, is important for the delay in diabetes development, the abnormal extravasation of IS-CD8+ cells in IFN-{gamma}-deficient mice could reflect a developmental defect of pancreatic endothelium. However, this is unlikely, as no visible morphological abnormalities have been detected in the pancreata of NOD.IFN-{gamma}-KO mice. Moreover, because the diapedesis of IS-CD8+ cells was partially corrected by the treatment of NOD.IFN-{gamma}-KO recipients with rIFN-{gamma} before the IS-CD8+ cells injection (Table IIGo), IFN-{gamma} deficiency is unlikely to cause an irreversible developmental defect in microcapillaries. Rather, it suggests that IFN-{gamma} is necessary to regulate signaling pathways specifically involved in diapedesis.

Although it is probable that the targets of IFN-{gamma} are endothelial cells of the microcapillaries, the source of the cytokine remains unknown. It can be produced systemically (and the fact that systemic delivery of IFN-{gamma} affected extravasation of IS-CD8+ cells in the IFN-{gamma}-deficient mice also suggested that systemic production could play a role), or locally. The existence and importance of the background systemic levels of IFN-{gamma} are supported by findings that mice with targeted disruption of the IFN-{gamma} inhibitor suppressor of cytokine function-1 develop severe autoimmunity reversible upon the disruption of IFN-{gamma} production (41).

Identification of the targets of IFN-{gamma} that regulate diapedesis of adherent lymphocytes is a matter of further investigation. Expression of several adhesion molecules has been shown to be regulated by IFN-{gamma} (42, 43, 44). Of those, the platelet endothelial cell adhesion molecule (PECAM; CD31) has been shown to be involved in diapedesis due to the homophilic interaction between the extracellular domains of PECAM on a leukocyte and on an endothelial cell (45). We have not found any difference in the steady state levels of expression of PECAM, mucosal addressin cell adhesion molecule, pNad, ICAM-1, and VCAM in NOD and NOD.IFN-{gamma}-KO mice by staining of the cryostat acetone-fixed pancreatic sections with the corresponding Abs (data not shown). Thus, participation of these molecules in the diapedesis of IS-CD8+ cells is unlikely.

Chemokines have been implicated in the regulation of diapedesis of lymphocytes in a tissue-specific manner (39, 46). Several endothelial chemokines have been demonstrated to be regulated by IFN-{gamma} (47, 48): CXCL10, CXCL9, CCL20, and others. However, the most compelling data implicating IFN-{gamma} as a controlling factor for diapedesis were reported for CCL5 (RANTES) (49, 50). Enhanced in vitro transmigration of Th1-type T cells was shown to be dependent on CCL5 produced by endothelial cells in response to IFN-{gamma} stimulation. Anti-CCL5 Ab blocked only transmigration, but not adherence, as did Ab against CCR5, one of three CCL5-binding receptors (CCR1, CCR3, and CCR5) (49). Whether CCL5 is involved in diapedesis of diabetogenic lymphocytes remains to be seen. IFN-{gamma} has also been shown to up-regulate inducible NO synthase expression in {beta} cells (51), which can lead to an increased local concentration of NO that can potentially affect endothelial permeability for activated T cells.

The results show that IFN-{gamma} production is required to provide an adequate environment for the function of diabetogenic T cells. Lack of IFN-{gamma} alters presentation of autoantigens by {beta} cells. Moreover, IFN-{gamma} affects extravasation of autoimmune T cells into the islets. Interestingly, in other experimental models of autoimmune diabetes, the absence of IFN-{gamma} or its receptor {alpha}-chain was shown to affect islet infiltration (15, 52). In mice with islet-specific expression of lymphocytic choriomeningitis virus glycoprotein or nucleoprotein (52), and in mice with transgenic expression of a TCR from a diabetogenic CD4+ T cell (15), the lack of IFN-{gamma} signaling led to abrogation of insulitis, but not periinsulitis, which in our terms would be described as accumulation at the islet entrance, or lack of diapedesis. Progression from periinsulitis to insulitis is a critical step in the development of diabetes. Several genetic studies (53, 54, 55) have shown that it is controlled by multiple loci. IFN-{gamma} as a pluripotent cytokine may be involved in regulation of the genes encoded by those loci.

Thus, in addition to other known roles of IFN-{gamma}, this cytokine is clearly able to contribute to the development of autoimmune diabetes by regulating the penetration of the islets by diabetogenic T cells.


   Acknowledgments
 
We thank Andrew Tcherepanov and Jeffrey Bedrozian for technical assistance, Drs. David Serreze and Edward Leiter for helpful discussions, and Dr. Karl W. Browman for help with statistical analysis of data.


   Footnotes
 
1 The work was supported by National Institutes of Health Grant IDDK53561, Juvenile Diabetes Research Foundation International Grant 527149, a core grant from The Jackson Laboratory (to A.V.C.), and a postdoctoral fellowship from Juvenile Diabetes Research Foundation International (to A.Y.S.). F.S.W is a Wellcome Trust Senior Research Fellow in Clinical Science. Back

2 Address correspondence and reprint requests to Dr. Alexander V. Chervonsky, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609. E-mail address: avc{at}jax.org Back

3 Abbreviations used in this paper: IDDM, insulin-dependent diabetes mellitus; DiI, didodecyl-tetramethylindocarbocyanine perchlorate; IS, insulin-specific; KO, knockout; LSD, least significant difference; NOD, nonobese diabetic; PECAM, platelet endothelial cell adhesion molecule. Back

Received for publication June 8, 2001. Accepted for publication September 28, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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