Recruitment and Activation of Macrophages by Pathogenic CD4 T Cells in Type 1 Diabetes: Evidence for Involvement of CCR8 and CCL1

Mice

NOD and NOD.scid breeding mice were initially acquired from The Jackson Laboratory or the Barbara Davis Center for Childhood Diabetes and were housed in specific pathogen-free conditions at the University of Colorado Health Sciences Center for Laboratory Animal Care. NOD.scid mice were housed in sterile isolation cages. Mice in NOD.scid litters (6–10 days old) were used as recipients in adoptive transfer experiments. Breeding mice and experimental animals were monitored for development of disease by urine glucose. The 6.9 TCR transgenic (TCR-Tg) mouse was produced using TCR genes from a diabetogenic T cell clone, BDC-6.9 (8). All procedures used were in accordance with institutional IACUC guidelines and approved by the UCHSC Animal Care and Use Committee.

Culture and expansion of T cell clones

T cell clones were established from spleen and lymph nodes of diabetic NOD mice (9, 10) and were restimulated every 2 wk with a β cell granule membrane fraction obtained from β cell tumors as a source of Ag (11), irradiated NOD spleen cells as APCs, and EL-4 supernatant as a source of IL-2 in complete medium (CM). CM is DMEM supplemented with 44 mM sodium bicarbonate, 0.55 mM l-arginine, 0.27 mM l-asparagine, 1.5 mM l-glutamine, 1 mM sodium pyruvate, 50 mg/L gentamicin sulfate, 50 μM 2-ME, 10 mM HEPES, and 10% FCS. Cell numbers were expanded for transfer experiments by subculturing 3–6 × 10⁶ T cells 4 days after restimulation in a 5-fold volume of CM and additional IL-2. T cells were harvested, washed 3 times, and resuspended in HBSS for injection into young (≤10 days of age) NOD.scid recipients.

Assessment of cytokines and chemokines in vitro by intracellular staining

Production of cytokines and chemokines by T cells in vitro was analyzed by intracellular cytokine staining, as described previously (6). In brief, T cells were stimulated in plates coated with 1 μg/ml anti-CD3 Ab for 24 h before Ab staining. The cells were surface stained in 50–100 μl of staining buffer (PBS, 0.5% BSA) containing rat anti-CD4 or isotype control Ab for 30–45 min, and then washed and fixed in 2% formaldehyde. Cells were resuspended in permeabilization buffer (staining buffer plus 0.5% saponin), containing an isotype control or specific Ab mix for intracellular cytokines/chemokines. Polyclonal intracellular staining Abs used were obtained from R&D Systems and included polyclonal goat IgG anti-CCL1 (TCA-3), anti-CCL3 (MIP-1α), anti-CCL4 (MIP-1β), anti-CCL5 (RANTES), anti-CCL6 (C10), anti-CCL9/10 (MIP-1γ), and anti-CCL21 (SLC) as primary Abs, followed by FITC-rabbit anti-goat secondary Ab (Vector Laboratories). Monoclonal digoxigenin-MTAC-2 anti-lymphotactin and Cy5-anti-digoxigenin secondary Abs were provided by B. Dorner (12).

Adoptive transfer of diabetes

For disease transfer experiments, expanded cell cultures were harvested and T cells (1 × 10⁷⁾ were injected i.p. into age-matched 6- to 10-day-old NOD.scid recipient mice. In some experiments, adoptive transfers were performed with spleen cells from diabetic NOD or TCR-Tg donors. Onset of diabetes was monitored by urine glucose screening and positive readings were confirmed by blood glucose measurement. Blood glucose concentrations >15 mM for more than 1 day were considered diagnostic of overt diabetes.

Recovery of cells from pancreas

Approximately 1 week after adoptive transfer with T cell clones or at onset of diabetes (4–6 wk) following diabetic spleen cell transfers, recipient NOD.scid mice were sacrificed. As per methods described previously (2, 6), spleens and pancreata were removed and digested in PBS containing collagenase and GolgiPlug (BD Biosciences) in a 37°C water bath for 20–40 min. Single-cell suspensions of the digested pancreata and spleens were prepared, flash-spun to remove tissue debris, and then centrifuged at 300 × g for 10 min at 4°C to pellet. Cells were resuspended in CM/GolgiPlug and incubated at 37°C for 3–5 h, and in the case of T cells, with or without PMA (100 ng/ml) and ionomycin (1 μg/ml).

Ex vivo analysis of cytokines, chemokines, and chemokine receptors

After culture ex vivo of cells isolated from the pancreas, cells were harvested and washed before resuspension in staining buffer/GolgiPlug. Cells were stained in a 96-well round-bottom plate, 2 wells for each set of Ab combinations, one for specific Ab mix, and one for isotype control mix. Abs for surface staining included anti-CD4, anti-CD11b, and anti-CCR5 (BD Biosciences); anti-F4/80 (Caltag); and anti-CXCR3 and anti-CCR8 (R&D Systems). Surface and intracellular cytokine staining was performed as described for in vitro intracellular cytokine/chemokine staining, with the modification of adding GolgiPlug to all reagents until fixation and preincubating in staining buffer containing 2 μg/ml FcBlock (BD Biosciences) for 5 min on ice before addition of surface staining Abs. Polyclonal intracellular staining Abs used were from R&D Systems and included polyclonal goat IgG, anti-CCL1 (TCA-3), anti-CCL3 (MIP-1α), anti-CCL4 (MIP-1β), anti-CCL5 (RANTES), anti-CCL6 (C10), anti-CCL9/10 (MIP-1γ), and anti-IL-1β as primary Abs, followed by FITC-rabbit anti-goat secondary Ab (Vector Laboratories) or Cy-5-conjugated donkey anti-goat secondary Ab (Jackson Immunoresearch). Staining for chemokine receptors was conducted without ex vivo culture on unfixed single-cell suspensions from pancreas or spleen as indicated.

Analysis of NO from macrophages stimulated by T cell clones

Resting T cells (8 × 10⁴) were cultured for 48 h in the presence of freshly isolated NOD thioglycolate-elicited peritoneal exudate cells (5 × 10⁴) as APC and 10 μg of a membrane preparation from β tumor cells as Ag. As an indirect assessment of NO, nitrite ion concentration in the harvested supernatants was measured and averaged from triplicate wells using the one-step Griess reagent assay (13) and compared with nitrite standards. The same medium used in the cell culture (CM) was the zero standard to account for nitrite background.

Macrophages are recruited to the pancreas by pathogenic CD4 T cells

To investigate the activity of CD4 T cell clones in vivo following adoptive transfer, pancreatic tissue was removed at various time points after transfer (but before onset of diabetes) and single cell suspensions of the infiltrating immune cells were analyzed by flow cytometry. We found that in addition to producing inflammatory cytokines in the pancreas, the pathogenic T cell clones recruited substantial numbers of macrophages, detectable at 2, 4, and 6 days after transfer (6). Similar data are illustrated in Fig. 1⇓ in a experiment in which the diabetogenic T cell clone BDC-2.5, or a nonpathogenic T cell clone BDC-2.4, were transferred to young NOD.scid recipients and a week later, cells were isolated from the pancreas and analyzed ex vivo for the presence of macrophages (Fig. 1⇓A). Comparison of the percentages of CD11b⁺ cells in pancreas after transfer of the two clones indicates that whereas substantial numbers of macrophages were observed in pancreas of mice receiving the BDC-2.5 T cell clone, the numbers of CD11b⁺ macrophages in pancreas of BDC-2.4 recipients were quite low, similar to those found in uninjected controls (data not shown). These results suggest that macrophage recruitment is linked with the pathogenic T cell response in autoimmune diabetes. Because CD11b is a marker for both macrophages and dendritic cells, we performed two-color staining on cells recruited to the pancreas for CD11b and for F4/80. Although expressed at lower levels on activated macrophages, the F4/80 Ag is commonly considered to be the most macrophage-specific marker (14). We found that almost all of the CD11b⁺ cells recruited to the pancreas also stained for the F4/80 Ag (Fig. 1⇓B), and therefore to identify macrophages in subsequent experiments, we used the brighter staining anti-CD11b Ab, which is a particularly valuable reagent in four-color staining from the pancreas ex vivo. It is possible, however, that the CD11b⁺F4/80⁺ cells may include some dendritic cells.

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FIGURE 1.

Macrophage recruitment to the pancreas by a pathogenic vs a nonpathogenic T cell clone. A, The percentage of CD11b⁺ cells (macrophages) in the pancreas was assessed at 6 to 7 days after transfer of a pathogenic T cell clone, BDC-2.5, or a nonpathogenic control, the Th1 T cell clone, BDC-2.4. Macrophage infiltration could be detected as early as 2 days after T cell clone transfer, but was considered optimal at 6 days. Because recipient mice start becoming diabetic at about 1 week after transfer, we did not go out to further time points. After removal and digestion of pancreata, cells were stained for the CD11b macrophage marker and analyzed by flow cytometry. Cumulative data from two separate experiments was used for the bar graph, and error bars represent SEM from 4 to 5 mice for each clone. ∗, p = 0.00015 × 2-tailed t test. B, F4/80 vs CD11b surface staining on pancreatic single-cell suspensions after transfer of BDC-2.5.

Macrophages recruited to the pancreas are activated to produce inflammatory cytokines

To test the hypothesis that macrophages are activated by diabetogenic T cells upon recruitment to the pancreas, we analyzed the CD11b-staining population recovered from the pancreas by intracellular staining. Our results showed that after transfer of the diabetogenic T cell clone BDC-2.5, large numbers of the infiltrating macrophages were producing TNF-α (Fig. 2⇓A). In contrast, adoptive transfer of the nonpathogenic clone BDC-2.4 resulted in few macrophages recruited and little to no stimulation of TNF-α production in the CD11b⁺ cells that were present. A second cytokine of interest in activated macrophages is IL-1β, a macrophage-specific cytokine that has been shown to be a very potent mediator of islet cell cytotoxicity (15, 16). As with TNF-α, we found that activation of macrophages to produce IL-1β was associated with disease induced by pathogenic CD4 T cell clones, and that there was a clear difference in macrophage IL-β levels in the pancreas after transfers with BDC-2.5 vs the nonpathogenic clone, BDC-2.4 (Fig. 2⇓B). These results suggest that production of inflammatory cytokines by macrophages is linked to the pathogenicity of CD4 T cells.

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FIGURE 2.

Inflammatory cytokine production in macrophages recruited to the pancreas by a pathogenic T cell clone. Expanded cultures of the BDC-2.5 T cell clone or the nonpathogenic BDC-2.4 Th1 T cell clone were injected into young NOD.scid recipients. At 4–6 days after transfer, pancreatic single-cell suspensions were incubated in the presence of Brefeldin A and were stained for surface expression of CD11b and for intracellular cytokine protein. TNF-α (A) or IL-1β (B) production by these macrophages was analyzed by flow cytometry. Histograms gated on CD11b⁺ cells are included to indicate the proportion of macrophages staining positive for the indicated cytokine protein. The data shown are representative of two independent experiments with similar results.

Activated macrophages in the pancreas also produce chemokines

We also wanted to know whether macrophages recruited to the pancreas after adoptive transfer of T cells were a significant source of inflammatory chemokines. We found that whereas CD11b⁺ cells isolated from the spleen produced very low levels of chemokines, there were substantial amounts of three chemokines found in macrophages recruited to the pancreas. Fig. 3⇓ shows intracellular staining in CD11b⁺ cells for CCL3 (MIP-1α), CCL4 (MIP-1β), and CCL6 (C10), 1 wk after transfer of the diabetogenic CD4 T cell clone BDC-2.5. As also illustrated in this figure, no staining was detected in macrophages for CCL1, CCL9/10, or CCL5. This result demonstrates that the macrophages recruited by pathogenic T cells are an important source of at least three inducible chemokines in the pancreas during disease.

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FIGURE 3.

Chemokine production in macrophages recruited to the pancreas by a pathogenic T cell clone. Six days following transfer of the BDC-2.5 clone into young NOD.scid recipients, single-cell suspensions of pancreas or spleen (pooled from 3 recipient mice) were incubated in the presence of Brefeldin A, stained for surface expression of CD11b and intracellular chemokine proteins, and analyzed by flow cytometry. Percentages in the upper right quadrants indicate the proportion of macrophages staining positive for the indicated chemokine. Chemokine staining in pancreatic macrophages in the left column is in contrast to the relative lack of chemokine expression in control splenic macrophages.

NO is another product of macrophages activated by diabetogenic CD4 T cells

NO can function either as an important mediator of positive cellular outcomes such as proliferation and activation, or as a signaling mediator leading to cell death (17). The deleterious effects of NO include its functioning as an intermediate in the production of reactive oxygen and reactive nitrogen species (ROS and RNS). The exact role of NO in T1D appears to be complicated (17), but its presence in combination with inflammatory cytokines, particularly IL-1β, is a strong predictor of islet-cell cytotoxicity (18, 19). Because macrophages are an important source of NO, we measured the ability of a panel of pathogenic CD4 T cell clones to stimulate NO production by macrophages, using the concentration of nitrite ions as an indirect measure of NO. By stimulating T cell clones with Ag, we are able to observe the effects on macrophages cocultured with the T cells and thus exposed to T cell-derived inflammatory cytokines. The data shown in Fig. 4⇓ show that the ability to stimulate macrophage production of this important inflammatory mediator is a property of diabetogenic CD4 Th1 T cells. In contrast, a Th2 T cell clone, 2.5Tg/T2-X, used as a control, was unable to stimulate NO production by macrophages, correlating with its inability to transfer disease to the NOD.scid mouse.

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FIGURE 4.

NO production by macrophages stimulated by diabetogenic T cell clones. Resting T cells (8 × 10⁴) were cultured for 48 h in the presence of freshly isolated NOD thioglycolate-elicited peritoneal exudate cells (5 × 10⁴) as APC and 10 μg of a membrane preparation from β tumor cells as Ag. As a measure of macrophage activation, nitrite ion concentration in the harvested supernatants was measured using the one-step Griess reagent assay, and compared with nitrite standards from duplicate wells. Results shown are obtained from cultures of six diabetogenic Th1 T cell clones and compared with data from a Th2 T cell clone (2.5 Tg/T2-X) used as a control. These data are representative of two identical experiments.

Recruitment and activation of macrophages is a manifestation of T cell effector function in other diabetes induction and spontaneous disease models

To determine whether macrophage recruitment and activation was a common feature of T1D pathogenesis, we analyzed macrophages isolated from the pancreas in several adoptive transfer and two spontaneous models of T cell-mediated autoimmune diabetes (Fig. 5⇓). Fig. 5⇓A shows inflammatory cytokine production by macrophages in two commonly used adoptive transfer systems involving spleen cells or in transfers with defined T cell clones. Spleen cell transfers included either heterogeneous populations of spleen cells from diabetic NOD mice or a quasi-clonal population of spleen cells obtained from the widely used BDC-2.5 TCR-Tg mouse. These systems were compared with transfers with two T cell clones, either a clone from our panel of diabetogenic CD4 T cell clones, or the pathogenic insulin-reactive CD8 T cell clone G9 described by Wong et al. (20). These different transfer models exhibit varying kinetics of disease induction in young NOD.scid recipients, but all depend on a population of pathogenic T cells to transfer disease. As a control, a CD4 Th2 T cell clone, 2.5Tg/T2-X, was used. The 2.5Tg/T2-X clone does not induce diabetes in NOD.scid recipients (21), nor does it lead to infiltration and cytokine production by macrophages (Fig. 5⇓B). In Fig. 5⇓C are results from analysis of macrophage inflammatory cytokine production in two spontaneous models of disease, either a spontaneously diabetic wild-type NOD mouse or a prediabetic 6.9 TCR-Tg/NOD.scid mouse (a TCR-Tg produced from a second diabetogenic CD4 T cell clone in our panel, BDC-6.9; see Ref. 8). Although mouse models for T1D differ in some respects and mechanisms of pathogenesis vary with the induction method, these results suggest that a common feature of disease mediated by pathogenic T cells, in both spontaneous and transfer models of diabetes, is a large number of infiltrating macrophages, many of which are producing inflammatory cytokines. The NOD spontaneous mouse model exhibits a longer time frame for disease onset, which likely explains the smaller numbers of macrophages in the pancreas at any given time point, even though a large percentage of them are producing inflammatory cytokine. Our findings lend support to the theory that the macrophage is a major effector cell for β cell destruction through its cytotoxic activity (22). The fact that this macrophage activity is dependent on pathogenic T cells is suggested by the lack of activated macrophages recruited to the pancreas by either a Th2 control clone (Fig. 5⇓B) or a nonpathogenic CD4 Th1 clone (Fig. 2⇑).

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FIGURE 5.

Recruitment and activation of macrophages seen in a variety of T1D mouse models. Single-cell suspensions from pancreas of recipients of adoptive transfers of pathogenic cells (A), recipients of a Th2 control clone (B), and spontaneously diabetic mice (C) were incubated in the presence of Brefeldin A and analyzed by flow cytometry for TNF-α or IL-1β production by CD11b⁺ macrophages. Analyses were performed 6–7 days after transfer of T cell clones or 3–4 wk after transfer of TCR transgenic or diabetic NOD spleen cells, as well as on pancreata from spontaneously diabetic adult NOD mice and prediabetic TCR transgenic mice (time points were chosen to be as close to disease onset as possible). Percentages in the upper right quadrants indicate the proportion of macrophages staining positive for TNF-α or IL-1β. Each dot plot represents the staining from an individual mouse and represents similar data from 2 to 3 mice for each transfer experiment.

Macrophages recruited to the pancreas express chemokine receptors

As illustrated in Fig. 1⇑, our results have demonstrated that adoptive transfer of diabetogenic CD4 T cell clones leads to the accumulation of large numbers of macrophages in the pancreas. For macrophages to be recruited to an inflammatory site in which T cells are producing cytokines and chemokines, they must express chemokine receptors. To investigate further the mechanisms by which macrophages are recruited to the pancreas during T1D, we examined the expression of three chemokine receptors that have been implicated in autoimmune diseases. CCR5 has been shown previously to be necessary for development of T1D (23) is a receptor for MIP-1α, MIP-1β, MIP-1γ, and RANTES, all of which are chemokines produced at the protein level by the pathogenic CD4 T cell clones in response to TCR stimulation in vitro (Table I⇓). Fig. 6⇓ shows that this receptor is expressed by virtually all CD11b⁺ macrophages recovered from the pancreas after transfer of BDC-2.5, whereas splenic macrophages were negative for CCR5. We also tested for expression of CXCR3 because this chemokine receptor was previously found to be required for infiltration into islets during progression to diabetes (24); however, the ligands for CXCR3 (CXCL9–11) were not expressed by the pathogenic T cell clones (J. Cantor and K. Haskins, unpublished data). As shown in Fig. 6⇓, CXCR3 is expressed at high levels on the surface of a substantial portion of CD11b⁺ macrophages recruited to the pancreas by BDC-2.5 and is also expressed at much lower levels on a portion of splenic macrophages. Possibly the most interesting finding from these experiments is that macrophages found in the pancreas after adoptive transfer of pathogenic T cells also express CCR8 and at levels that are much higher (>5-fold) than levels of CCR8 expression on splenic macrophages (Fig. 6⇓, bottom panels). Expression of CCR8 by recruited macrophages is of particular note because of the link between CCR8 and other autoimmune disease states (25, 32), and because it has not previously been studied in T1D.

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FIGURE 6.

Chemokine receptors on CD11b⁺ macrophages recruited to pancreas after transfer of a pathogenic CD4 T cell clone. Six days following transfer of the pathogenic CD4 Th1 T cell clone BDC-2.5 into young NOD.scid recipients, single-cell suspensions of pancreas or spleen were stained for surface expression of CD11b vs CCR5, CXCR3, or CCR8 and analyzed by flow cytometry. Filled peaks represent specific staining compared with isotype control Ab (unfilled peaks), gated on CD11b⁺ cells. Each histogram represents data from an individual mouse.

Diabetogenic T cells secrete numerous macrophage chemoattractants in vitro but only CCL1 in the pancreas

Chemokines are potent chemoattractants and possible activators of macrophages in inflammatory sites, and it has been reported that mRNA for several inflammatory chemokines can be detected in whole pancreas after disease transfer (26). We reported previously that a variety of chemokines can be detected both at the mRNA and at the protein level in a panel of seven CD4 diabetogenic T cell clones (6). Table I⇑ is a list of all the chemokines tested in three of the CD4 Th1 T cell clones; those that tested positive at the mRNA level were confirmed by intracellular staining. These results indicate that at least seven chemokines are made by diabetogenic T cells, all of which are documented chemoattractants for macrophages (27, 28, 29, 30, 31). In vitro, these chemokines are produced only upon TCR-mediated activation, with the exception of RANTES, a potent T cell chemokine that is present constitutively in unstimulated T cell clones.

Because macrophages and T cells migrating to the pancreas produce multiple inflammatory mediators, and as several chemoattractant receptors capable of binding multiple ligands are found on macrophages, it is not immediately apparent whether there is any one receptor-ligand pairing that is of particular significance in the developing disease process. Our results indicate that macrophages recruited to the pancreas express at least three chemokine receptors thought to be relevant to disease (Fig. 6⇑), the most interesting of which may be CCR8 because this chemokine receptor, unlike others, is known to bind only one ligand, the chemokine CCL1 (or TCA-3) (25). To further investigate the possible role of CCR8 on macrophages in T1D, we investigated whether diabetogenic CD4 T cell clones produce the ligand for this chemokine receptor in vivo. We adoptively transferred the T cell clone BDC-2.5 to NOD.scid mice and after removal spleen and pancreas 6 days later, we analyzed the T cells by intracellular staining for production of chemokines. Although in vitro the T cells can be induced to make several inflammatory chemokines, including CCL3 (MIP-1α), CCL4 (MIP-1β), CCL9/10 (MIP-1γ), CCL6 (C10), CCL5 (RANTES), and CCL1 (TCA-3) (6), the only chemokine that could be detected in CD4 T cells in the pancreas ex vivo was CCL1 (Fig. 7⇓). The few T cells that were found in the spleen were negative for all chemokines tested (data not shown). The production of CCL1 by pathogenic T cells in the pancreas, together with the expression of CCR8 on macrophages recruited to the pancreas, provide the first evidence that CCR8/CCL1 interaction may play a role in T1D.

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FIGURE 7.

CCL1 (TCA-3) production ex vivo by the pathogenic clone BDC-2.5 from the pancreas after transfer. Six days after transfer of the T cell clone BDC-2.5 into 9-day-old NOD.scid mice, single-cell suspensions of pancreas (pooled from 3 mice) were incubated in the presence of Brefeldin A, with or without PMA, and analyzed for intracellular staining by flow cytometry. Percentages in the upper right quadrants indicate the proportion of T cells staining positive for the indicated chemokine. A similar pattern of chemokine expression was seen upon repetition of this experiment.