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The Journal of Immunology, 2003, 170: 1649-1658.
Copyright © 2003 by The American Association of Immunologists

Th1 and Th2 Pancreatic Inflammation Differentially Affects Homing of Islet-Reactive CD4 Cells in Nonobese Diabetic Mice 1

Natasha J. Hill, Kurt Van Gunst and Nora Sarvetnick

The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The control of lymphocyte recruitment to the site of inflammation is an important component determining the pathogenicity of an autoimmune response. Progression from insulitis to diabetes in the nonobese diabetic mouse is typically associated with Th1 pancreatic inflammation, whereas Th2 inflammation can seemingly be controlled indefinitely. We show that a Th1 (IFN-{gamma}) pancreatic environment greatly accelerates the recruitment of adoptively transferred islet-specific CD4 T cells to the islets and also accelerates the onset of diabetes. The increased number of islet-reactive T cells in the pancreas does not result from increased proliferation or a decreased rate of apoptosis; instead, it appears to be caused by a greatly facilitated rate of entry to the pancreas. In contrast, a Th2 (IL-4) pancreatic environment does act to enhance Ag-specific proliferation and decrease the rate of apoptosis in islet-specific CD4 T cells. Nonpathogenic/regulatory cells are not preferentially expanded by the presence of IL-4. Increased recruitment to the islets was also observed in the presence of IL-4, but to a lesser extent than in the presence of IFN-{gamma}, and this lesser increase in the rate of recruitment did not accelerate diabetes onset within the time period examined. Therefore, the production of Th1 cytokines by initial islet-infiltrating cells may cause a greater increase than Th2 cytokines in the rate of recruitment of activated T cells. This difference in rate of recruitment may be critical in determining whether the initial infiltrate proceeds to diabetes or whether a steady state insulitis develops that can be maintained.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nonobese diabetic (NOD)3 mice spontaneously develop diabetes following the autoimmune destruction of {beta} cells in the islets. However, islet infiltration does not necessarily proceed to complete destruction of {beta} cells and diabetes. Instead, a nonpathogenic state of partial infiltration can be maintained indefinitely. Cytokines can alter the local expression of chemoattractant and adhesion molecules to increase recruitment of specific leukocyte populations, but it is not known whether homing to the pancreas is functionally affected by differential cytokine production. The production of Th1 cytokines such as IFN-{gamma} by initial islet-infiltrating cells may cause a greater increase in the subsequent rate of recruitment of activated T cells compared with the production of Th2 cytokines such as IL-4. This difference in rate of recruitment may be critical in determining whether the initial infiltrate proceeds to diabetes or whether a steady state insulitis develops that can be maintained. Partial infiltration and lack of progression to diabetes has been associated with the production of Th2 rather than Th1 cytokines by islet-infiltrating cells, for example in NOD.B10Idd9 congenic mice (1) and in NOD mice following administration of insulin (2, 3, 4), and also by restimulated splenocytes from islet autoantigen-treated NOD mice (5, 6, 7). The transition from nonpathogenic insulitis to diabetes during the natural course of disease in NOD mice also correlates with the change from a predominance of Th2 to Th1 cytokines in the islets (8). Furthermore, transgenic expression of IL-4 in the islets of NOD mice protects against diabetes (9).

The differential ability of Th1 and Th2 effector cells to migrate to the site of inflammation may be a critical determinant of their pathogenicity in autoimmune disease (10). Studies in the NOD mouse following adoptive transfer of in vitro differentiated Th1 and Th2 effector cells specific for islet Ag have shown that Th1 effector cells home to the pancreas more quickly and accelerate insulitis compared with Th2 effectors (11, 12, 13). The differential homing ability of Th1 and Th2 cells is attributed to the expression of partially distinct profiles of chemokine receptors and adhesion molecules (14, 15, 16).

Similarly, in a CD8 T cell-mediated model of type I diabetes using transfer of hemagglutinin-specific effector T cells into mice expressing hemagglutinin in the islets, the increased diabetogenicity of T cytotoxic (Tc)1 effector cells was caused by increased recruitment/accumulation in the pancreas compared with Tc2 effector cells because the two populations had equivalent cytotoxic activity in vitro (17). The importance of increased homing of Tc1 effector cells to the site of inflammation has also been demonstrated in models of viral clearance (18) and control of tumor growth (19) mediated by CD8 T cells. The differential homing capacities of Tc1 and Tc2 effector cells therefore contributes to their distinct functional abilities. However, CD8 lymphocytes activated in vivo in the presence of pancreatic IL-4 do have reduced cytotoxic activity, and this is mediated by altered dendritic cell Ag presentation (20).

A key function of inflammatory cytokines is to increase the recruitment of specific leukocyte populations into the site of inflammation (21). Tissue migration of leukocytes is controlled by three major types of interaction: selectins bind to their ligands to mediate initial tethering and rolling of leukocytes on endothelia, and chemokine receptor-ligand interactions then activate the next step of integrin binding to adhesion molecule receptors to allow entry into the tissue (21, 22, 23). Regulation of chemokine and adhesion molecule expression within an inflamed tissue by the production of Th1 or Th2 cytokines will preferentially recruit distinct sets of inflammatory cells (22). Islet-specific Th1 and Th2 cells stimulated in vitro express different patterns of chemokines, with Th1 cells expressing the T cell chemoattractants macrophage-inflammatory protein-1{alpha} and macrophage-inflammatory protein-1{beta} in particular at higher levels than Th2 cells (13). If Th1 cytokines more effectively recruit effector T cells to the pancreas compared with Th2 cytokines, this could cause the transition from a nonpathological insulitis that can be maintained to overt diabetes.

BDC2.5 CD4 T cells become activated and undergo proliferation in the pancreatic lymph nodes (panLN) before subsequent migration to the pancreas (24). However, it is not known how Th1 and Th2 effector cytokines produced in the pancreas affect the ongoing recruitment of naive CD4 T cells into the panLN or whether the potential of CD4 T cells activated in the panLN to home to the pancreas is also impacted. We wanted to examine whether the rate of lymphocyte recruitment, either at the level of naive T cell entry into the panLN or in the migration of activated effector T cells to the pancreas, is functionally affected by the presence of Th1 vs Th2 inflammation in the pancreas. To test this, we adoptively transferred naive islet-reactive CD4 (BDC2.5) T cells into IFN{gamma}NODScid, IL4NODScid, and control littermate NODScid recipients and compared the expansion and recruitment of injected cells to the panLN and to the pancreas.

We show that Th2 inflammation in the pancreas causes expansion of the islet-reactive CD4 population in the panLN, by increasing Ag-driven proliferation and lowering the rate of apoptosis, and moderately increases homing to the pancreas. In contrast, Th1 pancreatic inflammation does not increase islet-reactive CD4 cell numbers in the panLN but greatly accelerates migration to the pancreas. IFN{gamma}NODScid recipients develop diabetes 5 days after transfer of islet-reactive BDC2.5 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Adoptive transfer of BDC2.5 splenocytes

Six-week-old female NODBDC2.5 mice (originally provided by D. Mathis and C. Benoist (Harvard Medical School, Boston, MA)) were used in all experiments as donors of splenocytes for i.v. injection into IFN{gamma}NODScid, IL4NODScid, and transgene negative NODScid littermate recipients. Recipient mice were aged from 6 to 9 wk. In some experiments donor splenocytes were enriched using Stemsep magnetic separation columns (according to manufacturer’s instructions) to negatively select for CD4+ cells. Otherwise, unpurified cells were injected, and donor splenocytes were lysed in hypotonic solution to remove RBC. For CFSE labeling, cells were incubated at 5 x 107 cells/ml with 5 mM CFSE for 10 min at 37°C. The reaction was quenched with 10 ml of cold PBS, and the cells were washed once in PBS. The 5–8 x 106 CD4-enriched cells or 2 x 107 unpurified cells were injected in 200 µl of sterile PBS per recipient mouse. FACS staining determined that CD4-enriched cells were >98% CD4+V{beta}4+ (BDC2.5 TCR), and unpurified cells were ~20% CD4+Vb4+. In CFSE-labeling experiments 100% of the cells were CFSE positive.

Recovery of injected cells from panLN and spleen

At various timepoints following adoptive transfer, single cell suspensions were prepared from the spleen (data not shown) and panLN of recipients. The panLN cells were resuspended in 100 µl, and the whole sample was stained for flow cytometry. The total number of injected BDC2.5 cells present in the panLN was determined by staining with V{beta}4-biotin/SA-PerCP and CD4-APC Abs from BD PharMingen (San Diego, CA) and also with the appropriate isotype controls for analysis by flow cytometry. Dead cells were gated out on the basis of forward and side scatter. For analysis of CFSE dilution and CD69/CD62L activation markers (BD PharMingen), cells were gated on the CD4+V{beta}4+ population. The anti-BDC2.5 Ab was kindly provided by O. Kanagawa (Washington University School of Medicine, St. Louis, MO). For analysis of annexin, stain cells were gated on CD4+V{beta}4+7AAD- cells. All mean values quoted in the text are ± SEM.

Calculation of "precursor" number

The number of precursor BDC2.5 cells homed to the panLN was calculated by dividing the number of cells in each CFSE peak by 1/(2x) (where x is the number of cell divisions undergone by the cells in that peak) and taking the sum for all CFSE peaks (25). This gives an estimate of the number of CD4+V{beta}4+ cells homed to the panLN before expansion, assuming an equal rate of cell death. The sets of values for the IFN{gamma}NODScid and NODScid recipients, and the IL4NODScid and NODScid recipients were compared using the two-sample Student’s t test (two tailed).

Diabetes and insulitis score

For histological assessment of islet infiltration, pancreata were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections (4 µm) from two levels through the tissue, separated by at least 120 µm, were stained with either H&E or with anti-insulin Ab (DAKO, Carpinteria, CA; 10–15 ng/ml) and hematoxylin counterstain. Islets were scored as having no insulitis, peri-insulitis, insulitis, or severe insulitis (majority of islet destroyed).

For anti-CD4 (GK1.5 used at 2.5 ng/ml) and anti-V{beta}4 (KT4 used at 5 ng/ml) stain, the pancreas of recipient mice was quick-frozen in OCT, and 4-µm sections were cut for staining. Adjacent sections were also stained with an anti-insulin Ab and with the appropriate secondary Ab alone.

Blood glucose levels were monitored using Glucometer Elite strips. Mice with two successive blood glucose levels greater than 300 mg/dl were considered diabetic.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Recovery of injected BDC2.5 cells from the panLN of IL4NODScid and IFN{gamma}NODScid recipients

To test the impact of Th1 and Th2 pancreatic inflammation on the recruitment of naive T cells to the panLN, we injected IL4NODScid, IFN{gamma}NODScid, and transgene-negative littermate NODScid control mice with CFSE-labeled NODBDC2.5 splenocytes. Young NODBDC2.5 mice (6 wk of age) were used as donors; flow cytometry analysis of splenocytes from these mice showed that the BDC2.5 cells injected were 75–80% CD62L+CD44low and less than 5% CD25+ and therefore phenotypically naive (data not shown). We then took the panLN of recipient mice at days 3 and 4 following transfer and determined the number of BDC2.5 cells accumulated in the panLN by flow cytometry. The BDC2.5 population was identified by staining with Abs to CD4 and the BDC2.5 TCR, V{beta}4.

The mean recovery of injected BDC2.5 (CD4+V{beta}4+) cells from the panLN was increased by as much as 10-fold at day 4 in IL4NODScid recipients compared with NODScid recipients (Fig. 1, A and C, 3.4 x 104 ± 0.9 x 104, n = 3, and 3.9 x 103 ± 1.0 x 103, n = 5, respectively). In contrast, only a slight increase (~2-fold, 6.5 x 103 ± 1.5 x 103, n = 4, and 2.7 x 103 ± 0.6 x 103, n = 5, respectively) in the recovery of BDC2.5 cells from the panLN of IFN{gamma}NODScid recipients compared with NODScid recipients was observed at day 4 (Fig. 1, B and D). These data are representative of three independent experiments, although the increase in mean cell recovery in IFN{gamma}NODScid recipients at day 4 was not observed in one experiment.



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  		FIGURE 1. Recovery of BDC2.5 cells from the panLN is greatly increased in IL4NODScid recipients compared with NODScid recipients, but only slightly increased in IFN{gamma}NODScid recipients. The recovery of BDC2.5 cells from the panLN was determined by flow cytometry staining for CD4+V{beta}4+ cells on single cell suspensions prepared from IL4NODScid (A) and IFN{gamma}NODScid (B) recipients and their transgene negative littermates following adoptive transfer. Each recipient was injected with 2 x 107 CFSE-labeled splenocytes from 6-wk NODBDC2.5 mice. The mean number of BDC2.5 cells recovered from IL4NODScid (C) and IFN{gamma}NODScid (D) recipients and control littermates at the indicated timepoint is shown ± SEM; n = 3–6 for each strain at each timepoint. The transgenic and control mice in each experiment received cells from the same pooled donor splenocytes, but the IL4NODScid and IFN{gamma}NODScid experiments were performed at different times.

 
The number of BDC2.5 cells in the panLN at a particular timepoint will be the result of the relative rates at which several component factors occur, primarily the rate of naive cell recruitment to the lymph node, the rate of proliferation within the lymph node, the rate of apoptosis, and the rate of migration out of the lymph node following activation. Therefore, we assessed the role of each of these factors in this system to determine whether Th1 and Th2 inflammation differentially affect the rate of naive cell recruitment to the panLN.

Proliferation of BDC2.5 cells in the panLN of IL4NODScid and IFN{gamma}NODScid recipients

We first wanted to test whether the increased BDC2.5 population in IL4NODScid recipients could be explained by an increased rate of proliferation caused by the local production of IL-4. Therefore, the CFSE dilution profile of CD4+V{beta}4+-gated BDC2.5 cells in the panLN of each recipient strain was compared to determine the effect of pancreatic IL-4 and IFN-{gamma} production on the proliferation of naive BDC2.5 cells recruited to the panLN.

The CFSE profiles of injected BDC2.5 cells show that the number of cells proliferating in the panLN is greatly increased when IL-4 is present in the pancreas (Fig. 2A), but not in the presence of IFN-{gamma} (Fig. 2B). This can be observed at both days 3 and 4 following adoptive transfer. The proportion of undivided cells can be taken as an indication of the extent of proliferation, although this will also be influenced by the fraction of surviving progeny. The mean percentage of undivided cells is lower in IL4NODScid recipients than NODScid recipients at both day 3 (12 ± 1%, n = 4, and 36 ± 4%, n = 6, respectively) and day 4 (6 ± 1%, n = 3, and 17 ± 2%, n = 5, respectively). In contrast, the presence of IFN-{gamma} in the pancreas does not affect proliferation of BDC2.5 cells in the panLN (mean percentage of undivided cells in IFN{gamma}NODScid and NODScid recipients at day 3: 46 ± 2%, n = 3, and 42 ± 3%, n = 3, respectively; and day 4: 14 ± 3%, n = 4, and 21 ± 3%, n = 5, respectively).



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  		FIGURE 2. The number of BDC2.5 cells proliferating in the panLN is increased in IL4NODScid, but not IFN{gamma}NODScid, recipients compared with control NODScid recipients. The CFSE dilution profiles of CD4+V{beta}4+ cells in the panLN of the IL4NODScid (A) and IFN{gamma}NODScid (B) recipient mice injected as in Fig. 1 show that the expansion of BDC2.5 cells in IL4NODScid recipients is greater than in littermate controls.

 
Because we had injected cells into immunodeficient recipients, we wanted to test whether the increased expansion observed in IL4NODScid recipients was caused by the effect of IL-4 on Ag-specific proliferation rather than on homeostatic expansion. Therefore, we compared the proliferation of islet Ag-specific and nonspecific CD4 T cells in the panLN by injecting CFSE-labeled purified CD4 T cells from 6-wk-old NOD mice mixed in equal number with CFSE-labeled purified CD4 T cells from 6-wk-old NODBDC2.5 mice (5 x 106 CD4+ cells of each population). At day 3 following injection, panLN cells were taken for analysis by flow cytometry, and CD4+ cells were gated into V{beta}4+ or V{beta}4- populations. The CD4+V{beta}4+ (BDC2.5) population consists almost exclusively of islet Ag-specific cells, whereas the CD4+V{beta}4- population from NOD mice is expected to contain only a very low frequency of islet Ag-specific cells. The NOD spleen will also contain a CD4+V{beta}4+ population that is not necessarily islet specific, but this will again be a low percentage compared with the large population of BDC2.5 V{beta}4+ islet-specific CD4 cells.

We then compared the CFSE dilution profiles of the islet-specific (V{beta}4+) and nonspecific (V{beta}4-) populations in the panLN of IL4NODScid, IFN{gamma}NODScid, and control NODScid recipients at day 3 following transfer. Proliferation in the panLN of IL4NODScid (Fig. 3A), IFN{gamma}NODScid (Fig. 3B), and control NODScid recipients occurs almost exclusively in the CD4+V{beta}4+ population (n = 2). On average, 83 ± 2% (n = 8) of the CD4+V{beta}4- population was undivided in the three recipient strains (the results for the CD4+V{beta}4+ population in each strain were comparable with the results for day 3 reported above). Therefore, at the early timepoints examined, proliferation occurs only in islet-specific CD4 T cells.



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  		FIGURE 3. Proliferation of injected CD4 cells in panLN is islet Ag specific. CD4-enriched CFSE-labeled cells from 6-wk NODBDC2.5 donors and 6-wk NOD donors were mixed in equal proportion (5 x 106 cells of each population) and injected into IL4NODScid (A) and IFN{gamma}NODScid (B) recipients. At day 3 following injection, panLN cells were taken for flow cytometry, and CD4+ cells were gated into V{beta}4+ or V{beta}4- populations. The CD4+V{beta}4+ population will consist largely of islet Ag-specific BDC2.5 cells, whereas the CD4+V{beta}4- population will contain only a low frequency of islet-specific cells. Expansion occurs almost exclusively in the V{beta}4+ population. Therefore, the proliferation observed is islet Ag specific. n = 2 for each strain. Proliferation of BDC2.5 cells in the panLN of immunodeficient recipients is also associated with the expression of activation markers up-regulated during Ag-specific, but not homeostatic, expansion. CFSE-labeled cells from 6-wk NODBDC2.5 donors were transferred to IL4NODScid and NODScid recipients and panLN cells taken at day 3 for analysis of CD69 expression by CD4+V{beta}4+ BDC2.5 cells (C). The percentage expression of CD69 in BDC2.5 cells of each CFSE dilution peak is plotted (mean values ± SEM, n = 3 for each strain). The data is representative of two independent experiments.

 
We also examined expression of the early activation marker CD69 that has been shown to be up-regulated during Ag-specific, but not homeostatic, expansion (26, 27). CFSE-labeled NODBDC2.5 splenocytes were transferred into IL4NODScid and NODScid recipients, and the expression of CD69 in each CFSE-dilution peak of the CD4+V{beta}4+ BDC2.5 population in the panLN at day 3 following transfer was determined. In both IL4NODScid and NODScid recipients, expression of CD69 is up-regulated on BDC2.5 cells as they divide (Fig. 3C). After three rounds of division almost 60% of CD4+V{beta}4+ cells express CD69 in both strains (n = 3 for each strain, and the results are representative of two independent experiments). In IL4NODScid recipients, the mean expression of CD69 in the total BDC2.5 population is 31 ± 1% and in NODScid recipients 24 ± 3%. In IL4NODScid recipients, CD69 expression is also slightly increased in undivided BDC2.5 cells and in cells that have undergone 1–2 rounds of division, suggesting that activation of islet-reactive cells is increased by the presence of pancreatic IL-4. Similar results were also observed for the down-regulation of CD62L on proliferating BDC2.5 cells (data not shown).

Exclusion of the TCR {beta}-chain is complete in donor transgenic NODBDC2.5 mice, but exclusion of the {alpha}-chain is incomplete (28). Spontaneous diabetes in NOD mice is accelerated when the BDC2.5 TCR is expressed on a background that inhibits rearrangement of endogenous TCR, either by introducing the Scid (29), Rag, or C{alpha} mutations (30). Therefore, it has been proposed that NODBDC2.5 mice contain a population of nonpathogenic/regulatory cells that can suppress the onset of diabetes (30, 31). A recently described Ab specifically recognizes clonotype-positive BDC2.5 cells. CD4 cells from NODBDC2.5 mice that are recognized by this anti-BDC Ab efficiently transfer diabetes to NODScid recipients, whereas those that are not recognized by this Ab do not transfer diabetes and when mixed in equal number can suppress diabetes induced by anti-BDC positive cells (31). Therefore, we used this Ab to test whether the nondiabetogenic/regulatory clonotype-negative population is preferentially expanded in IL4NODScid recipients or deleted in IFN{gamma}NODScid recipients.

The panLN cells from recipient mice at day 3 following transfer of CFSE-labeled NODBDC2.5 splenocytes were stained with Abs to CD4 and with the anti-BDC Ab. Only CD4+aBDC+ cells in IL4NODScid recipients showed the expansion observed previously in the CD4+Vb4+ population in this strain in terms of cell number and CFSE profile (Fig. 4A). Furthermore, the percentage of CD4 T cells not recognized by the aBDC Ab is actually reduced in IL4NODScid compared with NODScid recipients because of the greater expansion of aBDC+ cells (Fig. 4B). Therefore, the presence of IL-4 specifically expands the diabetogenic BDC2.5 clonotype-positive population and not nonpathogenic/regulatory cells expressing endogenously rearranged TCR. The CD4+aBDC- population was present at a comparable cell number and showed a similar CFSE profile in IFN{gamma}NODScid and NODScid recipients (data not shown). Therefore, this population is not deleted in IFN{gamma}NODScid recipients.



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  		FIGURE 4. CD4 T cells expressing the BDC2.5 clonotype are expanded in IL4NODScid recipients but not in nonpathogenic CD4 T cells not expressing the clonotype BDC2.5 TCR. Cells from the panLN of recipient mice injected as in Fig. 1 were additionally stained at day 3 with an Ab specific for the BDC2.5 clonotype (aBDC) that distinguishes between diabetogenic BDC2.5 cells (aBDC+) and nonpathogenic/regulatory cells expressing endogenously rearranged V{alpha}-chains (aBDC-). The CFSE profile (A) of CFSE+CD4+ cells in IL4NODScid and NODScid recipients gated into aBDC+ and aBDC- populations is shown in A, and the percentage of CFSE+CD4+ cells recognized by the aBDC Ab is shown in B. The data shown is representative of two experiments, n = 4–5 for each recipient strain.

 
These experiments demonstrate that the proliferation of islet-specific CD4 T cells in the panLN of IL4NODScid recipients is increased compared with NODScid recipients. IL-4 does not induce the expansion of nonpathogenic/regulatory CD4 cells with endogenously rearranged TCR. Only islet Ag-specific cells proliferate, and expansion is associated with up-regulation of the activation marker CD69 that is specifically expressed during Ag-driven, and not homeostatic, proliferation. However, whereas the expansion observed is Ag specific, it is likely that the rate of Ag-driven expansion is being enhanced by the immunodeficient environment of the recipients and that the presence of IL-4 further potentiates this homeostatically enhanced islet-driven expansion.

Calculation of the number of precursor BDC2.5 cells homed to the panLN

To quantitatively compare the number of BDC2.5 cells recruited to the panLN independently of the different rates of expansion in IL4NODScid and NODScid recipients, we calculated the precursor number of BDC2.5 cells in the panLN using the CFSE dilution and absolute BDC2.5 cell number data described above. The precursor number represents an estimate of the number of BDC2.5 cells recruited to the panLN before division and is determined using the number of cells in each CFSE division peak corrected for the number of divisions that have occurred (see Materials and Methods). From these calculations it can be seen that the mean BDC2.5 precursor number in IL4NODScid recipients is increased ~5-fold compared with NODScid recipients (Fig. 5A). This is consistent at both day 3 (3.6 ± 0.8 x 103 and 0.7 ± 0.1 x 103, respectively, p < 0.01) and day 4 (4.3 ± 0.8 x 103 and 0.7 ± 0.2 x 103, respectively, p < 0.005) following transfer.



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  		FIGURE 5. The calculated number of CD4+V{beta}4+ precursor cells homed to the panLN is increased in IL4NODScid recipients, and slightly increased by day 4 in IFN{gamma}NODScid recipients, compared with control NODScid recipients. Using the CFSE dilution profiles of BDC2.5 T cells in the panLN of IL4NODScid (A) and IFN{gamma}NODScid (B) recipient mice, the number of precursor BDC2.5 cells homed to the panLN was calculated (see Materials and Methods). This gives an estimate of the number of CD4+V{beta}4+ cells homed to the panLN before expansion. A horizontal line indicates the mean value. The transgenic and control mice in each experiment received cells from the same pooled donor splenocytes, but the IL4NODScid and IFN{gamma}NODScid experiments were performed at different times. The cell recovery on day 3 of the IFN{gamma}NODScid experiment shown is low, but the lack of difference between the IFN{gamma}NODScid and control precursor number is consistently observed. The precursor number will also be affected if the presence of IFN-{gamma} or IL-4 in the pancreas alters the rate of apoptosis in the BDC2.5 population. Therefore, it was important to examine the rate of apoptosis in the CD4+V{beta}4+ population in the three recipient strains.

 
Similar calculations comparing the precursor number in IFN{gamma}NODScid and NODScid recipients (Fig. 5B) show that no difference can be seen at day 3 (160 ± 36 and 197 ± 70, respectively), although a modest increase of ~2-fold can be seen at day 4 (1.7 ± 0.5 x 103 and 0.8 ± 0.2 x 103, respectively), although this increase was not observed in one of three experiments performed.

The increased precursor number in IL4NODScid, but not IFN{gamma}NODScid, recipients suggests that recruitment to the panLN may be enhanced by the presence of IL-4 in the pancreas, whereas IFN-{gamma} does not significantly alter recruitment to the panLN. However, the precursor number of BDC2.5 cells in the panLN will also be influenced by any differences in the rate of cell death. Therefore, we examined the rate of apoptosis of BDC2.5 cells in the panLN in recipient strains.

The rate of apoptosis in BDC2.5 cells in the panLN of IL4NODScid and IFN{gamma}NODScid recipients

To test whether the rate of apoptosis in BDC2.5 cells recruited to the panLN is decreased by the presence of IL-4 in the pancreas or increased by the presence of IFN-{gamma}, we again injected splenocytes from young NODBDC2.5 donors into IFN{gamma}NODScid, IL4NODScid, and NODScid littermates. At day 3 following transfer, panLN cells were stained with anti-CD4, anti-V{beta}4, annexin V, and the live cell dye 7AAD for analysis by flow cytometry. The percentage of BDC2.5 cells undergoing apoptosis wasmeasured as the percentage of annexin-positive cells in the live (7AAD negative) CD4+V{beta}4+ gated population (Fig. 6A).



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  		FIGURE 6. The rate of apoptosis in BDC2.5 cells is decreased in IL4NODScid recipient mice. At day 3 following transfer of NODBDC2.5 splenocytes, cell suspensions from panLN of recipients were stained for analysis by flow cytometry. Cells were gated on the 7AAD-negative, CD4+V{beta}4+ population (A). The percentage of annexin-positive cells in this gate was determined in IL4NODScid, IFN{gamma}NODScid, and control NODScid recipient mice (B). The mean percentage of annexin+ cells for each strain is shown on the histograms. The percentage of annexin stain for individual mice is plotted in C. The data are representative of two independent experiments.

 
The rate of apoptosis in BDC2.5 cells recruited to the panLN is significantly lower in IL4NODScid (2.3 ± 0.2% of CD4+V{beta}4+ cells annexin positive, n = 4) than NODScid recipients (9.1 ± 1.4% of CD4+V{beta}4+ cells annexin positive, n = 7; p < 0.01), as shown in Fig. 6, B and C. This decreased rate of death could account for the increased precursor cell number observed in IL4NODScid mice, rather than there being an increase in the recruitment of BDC2.5 cells to the panLN in the presence of IL-4. No significant difference in the percentage of annexin-positive BDC2.5 cells was observed between IFN{gamma}NODScid (15.6 ± 3.6% of CD4+Vb4+ cells annexin positive, n = 4) and NODScid recipients, although a slight increase in the mean percentage of annexin-positive cells was observed. Therefore, the presence of IFN-{gamma} in the pancreas does not affect the rate of apoptosis of adoptively transferred BDC2.5 cells in the panLN.

Homing of BDC2.5 cells to the pancreas in IFN{gamma}NODScid and IL4NODScid recipients

Using the same adoptive transfer protocol, we also wished to test whether the homing of injected BDC2.5 cells to the pancreas was affected by the presence of IFN-{gamma} and IL-4.

At day 4 following transfer of splenocytes from young NODBDC2.5 donors, the pancreas from IL4NODScid, IFN{gamma}NODScid, and NODScid recipient mice was fixed for histological analysis to assess the extent of islet infiltration in each strain by H&E and insulin staining of pancreatic sections.

Homing of injected cells to the pancreas was greatly increased in IFN{gamma}NODScid recipients compared with NODScid recipients, as shown in Fig. 7. It was not possible to score insulitis in the conventional way for IFN{gamma}NODScid recipients because of the unusual morphology in the pancreas of these mice and also because at day 4 following transfer the extent of islet destruction was such that only very few insulin-positive cells could be observed. The pancreas of IFN{gamma}NODScid mice before transfer of cells shows the characteristic intraductal islets and morphology typical of mice expressing IFN-{gamma} in the pancreas (Fig. 7, A and B). The recruitment of nonlymphocyte cells can also be seen in untreated IFN{gamma}NODScid mice, although diabetes does not occur in these mice. However, from the vast infiltration of lymphocytes and almost complete loss of insulin-producing cells by day 4 following transfer of BDC2.5 cells (Fig. 7, C and D), it is evident that homing to the pancreas is much increased compared with the limited, largely peri-insulitis that is observed in NODScid recipients at this time (Fig. 7, E and F). Therefore, whereas the presence of IFN-{gamma} in the pancreas has little effect on the homing or proliferation of BDC2.5 cells in the panLN, recruitment to the pancreas is greatly accelerated.



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  		FIGURE 7. Homing to the pancreas is greatly accelerated in IFN{gamma}NODScid recipients and moderately accelerated in IL4NODScid recipients. Paraffin sections from IFN{gamma}NODScid recipients that were either not injected with any cells (A and B) or at day 4 following transfer of unpurified BDC2.5 splenocytes (C and D), and also from day 4 NODScid (E and F) and IL4NODScid (G and H) recipients. Adjacent sections were stained with H&E (A, C, E, and G) or with anti-insulin/hematoxylin (B, D, F, and H).

 
Homing to the pancreas was moderately accelerated in IL4NODScid islets (Fig. 7, G and H) compared with NODScid islets; >40% of islets have insulitis/severe insulitis in IL4NODScid recipients 4 days following transfer, whereas only 20% of NODScid recipients have insulitis/severe insulitis at this time, and 60% of NODScid islets are free of infiltrating cells compared with 30% in IL4NODScid recipients (Fig. 8).



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  		FIGURE 8. Insulitis scoring in IL4NODScid and NODScid recipients. H&E-stained sections from IL4NODScid and NODScid recipients at day 4 following transfer were scored for insulitis as indicated. Sections were taken from two different levels through the pancreas separated by at least 120 µm. A total of 332 islets from 17 NODScid recipients and 296 islets from 10 IL4NODScid recipients were scored.

 
These data imply that an islet infiltrate producing predominantly IL-4 will increase recruitment of islet-reactive CD4 cells to the pancreas, but infiltration will be increased to a much greater extent if the islet-infiltrating cells are producing IFN-{gamma}.

It has previously been shown that adoptively transferred BDC2.5 cells appear first in the panLN and become activated, and only at later timepoints appear in the pancreas (24). We have shown that only the islet-specific BDC2.5 cells undergo proliferation in the panLN at the early timepoints examined (Fig. 3). Ag-specific activation of lymphocytes is known to result in the regulated expression of a program of adhesion molecules and chemokine receptors that will redirect the newly activated cell to the appropriate effector compartment. Therefore, it would be predicted that only the islet Ag-activated BDC2.5 cells within the panLN would be recruited to the pancreas. We tested this by staining sections of pancreas from recipient mice injected with a mixture of equal numbers of CD4-purified splenocytes from NODBDC2.5 and NOD mice, as described above, with Abs to CD4 and V{beta}4. As shown in Fig. 9, in IFN{gamma}NODScid (A–D), NODScid (E–H), and IL4NODScid (I–L) recipients the CD4 and V{beta}4 Abs appear to stain overlapping areas, and both Abs stained the infiltrate extensively. Therefore, the recruitment of CD4 cells to the islets appears to be specific for BDC2.5 cells and not nonislet Ag CD4 cells. This supports the idea that Ag-specific activation of CD4 cells in the panLN is a prerequisite for subsequent recruitment to the pancreas.



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  		FIGURE 9. Homing to the pancreas is specific to transferred islet-specific BDC2.5 cells. The pancreata of mice that had been injected with equal numbers of islet-specific NODBDC2.5 splenocytes and nonspecific NOD splenocytes (described in Fig. 3) were frozen in OCT medium, and adjacent 4 µm sections were stained with Abs to insulin (A, E, and I), CD4 (B, F, and J), and V{beta}4 (C, G, and K), and with the relevant secondary Ab alone (D, H, and L). Pancreata from IFN{gamma}NODScid (A–D), NODScid (E–H), and IL4NODScid (I–L) are shown.

 
Diabetes development in IFN{gamma}NODScid, IL4NODScid, and NODScid recipient mice

To test whether the difference in rate of recruitment resulting from the presence of IL-4 and IFN-{gamma} in the pancreas correlates with the progression of diabetes in this adoptive transfer model, we measured blood glucose in IFN{gamma}NODScid, IL4NODScid, and NODScid recipients of NODBDC2.5 splenocytes. As shown in Fig. 10, diabetes was greatly accelerated in IFN{gamma}NODScid recipients, and 100% of IFN{gamma}NODScid recipients (n = 11) develop diabetes by day 5 following transfer, whereas no IL4NODScid (n = 25) or NODScid recipients (n = 28) were diabetic at this time. Therefore, the greater rate of recruitment of islet-reactive cells to the IFN-{gamma} pancreas is associated with an acceleration of diabetes onset that does not occur following the lesser increase in recruitment in the presence of IL-4.



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  		FIGURE 10. IFN{gamma}NODScid recipients develop accelerated diabetes 5 days following adoptive transfer. Unpurified BDC2.5 splenocytes were adoptively transferred into IFN{gamma}NODScid (n = 11), IL4NODScid (n = 25), and transgene-negative littermate control NODScid (n = 28) mice, and blood glucose levels were monitored daily following transfer.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We show that the presence of a Th2 (IL-4) pancreatic environment dramatically increases the number of islet-reactive cells in the panLN and that this is largely explained by enhanced Ag-driven proliferation and a decreased rate of apoptosis. In contrast, a Th1 (IFN-{gamma}) pancreatic environment does not alter the size of the CD4 population in the panLN, and no significant change in proliferation or in the rate of apoptosis was observed in IFN{gamma}NODScid recipients.

However, a pancreatic Th1 cytokine environment greatly accelerates the recruitment of islet-specific CD4 cells to the pancreas. The presence of a Th2 cytokine environment also increases homing of islet-specific CD4 cells to the pancreas, but to a lesser extent compared with a Th1 cytokine environment. The greater acceleration of recruitment to the pancreas in the presence of IFN-{gamma} is associated with a rapid onset of diabetes that is not observed with the lesser acceleration in the presence of IL-4. Therefore, production of Th1 and Th2 cytokines in the pancreas results in differential recruitment of islet-reactive cells, and only the greatly increased rate of recruitment in the presence of IFN-{gamma} is associated with the early onset of diabetes.

In contrast to previous studies that have compared the homing of in vitro differentiated Th1 and Th2 effector cells to the pancreas, we injected naive islet-reactive CD4 cells into recipients expressing either the Th1 cytokine IFN-{gamma} or the Th2 cytokine IL-4 in the pancreas. In this way we could compare the effect of an established cytokine environment on the further recruitment of naive islet-reactive lymphocytes to test our hypothesis that the production of Th1 cytokines by the islet infiltrate causes a more rapid recruitment of T cells to the pancreas and that this is important in the transition from a partial insulitis that can be maintained and total {beta} cell destruction. Katz et al. (12) used splenocytes from NODBDC2.5/C{alpha}° mice differentiated in vitro into either Th1 or Th2 effector cells to inject neonatal NOD mice. Their results showed that initial recruitment of Th1 and Th2 effector cells to the islets was equivalent, and both populations could invade the islets. However, following injection of Th2 effector cells, progression to complete {beta} cell destruction did not occur, whereas the injection of Th1 effector cells resulted in diabetes starting at 7 days following transfer. By extension from our data, the lack of progression to diabetes following Th2 transfer could be caused by less efficient recruitment of new lymphocytes because of the production of Th2 rather than Th1 cytokines in the pancreas by the initial infiltrating cells. We were also able to examine the effect of Th1 and Th2 cytokine environments on the proliferation and apoptosis of islet-reactive cells in the panLN and to distinguish changes in these parameters from differences in naive T cell recruitment. Our results imply that an important component of the increased diabetogenicity of islet-infiltrating cells producing Th1 rather than Th2 cytokines is through increased recruitment of islet-specific lymphocytes to the pancreas rather than through increased expansion of the islet-reactive CD4 population in the pancreas.

We have focused on events at early timepoints following transfer and have not examined whether IL4NODScid recipients develop diabetes at a later time or whether they will be protected. We show that IL-4 primarily enhances islet-specific expansion and does not expand nonpathogenic/regulatory CD4 T cells. It is possible that the expansion of islet-reactive T cells in the panLN of IL4NODScid recipients represents a transient phenomenon that is later followed by increased apoptosis and a contraction of the islet-reactive population in the panLN and would not confer anyincreased risk of diabetes. However, we do observe insulitis in IL4NODScid recipients following transfer of BDC2.5 splenocytes, whereas IL4NOD mice are protected against spontaneous diabetes (9). The immunodeficient environment of IL4NODScid recipients may contribute to the development of insulitis, and it has recently been shown that nonpathogenic Th2 cells can induce autoimmunity following transfer to immunodeficient recipients (32, 33, 34, 35). It has previously been observed that IL-4 can enhance homeostatic proliferation of naive CD8 T cells (36), and our data support the hypothesis that IL-4 can increase homeostatically enhanced Ag-driven proliferation of naive CD4 T cells. There is also evidence suggesting that IL-4 has limited capacity to inhibit (12), and may even enhance, the diabetogenic potential of BDC2.5 T cells. IL4NODBDC2.5 double-transgenic mice develop diabetes (37), and IL4NODBDC2.5 splenocytes have increased Ag presentation function compared with NODBDC2.5 splenocytes. Furthermore, IL4NODBDC2.5 T cells, but not NODBDC2.5 T cells, develop a Th1 phenotype (38). Therefore, pancreatic expression of IL-4 may enhance the diabetogenicity of BDC2.5 T cells in the absence of significant numbers of T cells of other specificity, although in the time scale examined we do not observe the accelerated diabetes in IL4NODScid recipients that occurs in IFN{gamma}NODScid recipients.

Expansion of the islet-reactive CD4 population in the panLN of IL4NODScid recipients is linked to a moderate increase in homing to the islets compared with NODScid recipients. Up-regulation of adhesion molecules, chemokines, or selectins in the pancreas may underlie the moderate acceleration of insulitis seen in IL4NODScid recipients compared with NODScid recipients at the early timepoint examined. Alternatively, the acceleration of insulitis may directly reflect the expanded islet-reactive T cell population in the panLN of IL4NODScid recipients. However, this expansion of the panLN population was not required for the very much greater increased homing to the islets and acceleration of diabetes in IFN{gamma}NODScid recipients. One rationale for this is that IL-4 may promote retention of activated T cells in the panLN to orchestrate B cell help and humoral responses. Activated T cells specialized for B cell help express CXCR5 to localize to B cell follicles and lack P-selectin ligand expression (39, 40, 41, 42). In contrast, IFN-{gamma} may promote the rapid exit of newly activated T cells from the lymph node and target a higher proportion of effector cells to the pancreas, possibly by increasing expression of P-selectin ligand. T cells infiltrating the pancreas have been observed to express P-selectin ligand (43). Therefore, in future experiments it will beimportant to examine expression of chemokine receptors and P-selectin ligand on BDC2.5 cells in the panLN of IL4NODScid and IFN{gamma}NODScid recipients and to examine the panLN of IL4NODScid recipients histologically for evidence of increased germinal center formation.

Although the effect of IL-4 in the pancreas appears primarily to affect expansion of the islet-specific population in the panLN, the main effect of pancreatic IFN-{gamma} revealed in these studies is to increase recruitment of islet-specific cells to the pancreatic islets. It has previously been shown that IFN{gamma}BALB/c transgenic mice have increased expression of mucosal addressin cell adhesion molecule-1 within islets and on the surrounding acinar tissue and increased expression of ICAM-1 on pancreatic ductal epithelial cells and endothelial cells from an early age (44). IFN-{gamma} is known to up-regulate chemokines such as IFN-{gamma}-inducible protein-10 (CXCL10), monokine induced by IFN-{gamma} (CXCL9), and IFN-inducible T cell {alpha} chemoattractant (CXCL11), and these are chemotactic for activated T cells (45, 46, 47). The importance of IFN-{gamma} in homing of diabetogenic lymphocytes is supported by the results of recent experiments using IFN-{gamma} knockout mice showing that IFN-{gamma} is important in allowing diabetogenic CD8 T cells to enter the islets (48).

In these experiments we have not addressed whether the balance of differentiation into Th1 or Th2 effector cells is altered in IFN{gamma}NODScid and IL4NODScid recipients. However, Th1 and Th2 effector cells are known to express distinct patterns of chemokine receptors, with the CXCR3+CCR4- population consisting predominantly of Th1 cells and the CXCR3-CCR4+ population consisting predominantly of Th2 cells (15, 16, 49). The chemokine ligands for CXCR3 include the IFN-{gamma}-inducible protein-10, monokine induced by IFN-{gamma}, and IFN-inducible T cell {alpha} chemoattractant (50). The chemokine ligands for CCR4 include the IL-4/IL-13-induced chemokines macrophage-derived chemokine and thymus- and activation-regulated chemokine (51, 52). It has been reported that Th1 but not Th2 cells express functional P-selectin ligand (14) and can migrate in response to E- and P-selectins (53). It will be possible to address in vivo the question of whether a Th1 environment preferentially attracts Th1 rather than Th2 effector cells, and vice versa, by transferring fluorescently labeled Th1/Th2 effector cells into IFN{gamma}NODScid and IL4NODScid recipients and quantifying the relative migration by confocal imaging of pancreatic sections.

The results presented here in conjunction with future studies will provide a greater understanding of how cytokine production in the pancreas during ongoing inflammation affects the dynamics of effector cell recruitment to the pancreas and the progression of insulitis to diabetes.


   Acknowledgments
 
We thank Osami Kanagawa for providing the anti-BDC2.5 Ab, Mary Cleary and Lee Tucker for technical assistance, members of the laboratory for review of the manuscript and general discussions, and Marcia McDuffie for helpful comments and suggestions.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant DK55230. Back

2 Address correspondence and reprint requests to Dr. Nora Sarvetnick, The Scripps Research Institute, 10550 North Torrey Pines Road, IMM-23, La Jolla, CA 92037. E-mail address: noras{at}scripps.edu Back

3 Abbreviations used in this paper: NOD, nonobese diabetic; panLN, pancreatic lymph node; Tc, T cytotoxic. Back

Received for publication August 8, 2002. Accepted for publication December 2, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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