1 Division of Endocrinology and Diabetes, Department of Medicine, Keck School of Medicine,University of Southern California, Los Angeles, CA 90033, USA 2 Departments of Medicinal & Biological Chemistry and Pharmacology, College of Pharmacy,University of Toledo, Toledo, OH 43606, USA
Correspondence to: H. von Grafenstein, Department of Medicinal & Biological Chemistry, College of Pharmacy, University of Toledo,2801 West Bancroft Street, MS #606, Toledo, OH 43606, USA. E-mail: hgrafen{at}utnet.utoledo.edu
Transmitting editor: T. Watanabe
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Abstract |
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Keywords: cellcell interaction, ICAM-1, LFA-1, NO, type 1 diabetes
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Introduction |
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Activated macrophages release oxidants, including NO, peroxide, cytokines and inflammatory mediators that are, alone or in combination, toxic to ß cells (8) or other targets of destruction. However, because macrophages do not possess receptors that specifically recognize antigens expressed by targets such as ß cells, it is not clear how macrophage activation and macrophage-mediated tissue destruction are linked to recognition of target antigens.
T cells, on the other hand, can recognize antigens through highly specific antigen receptors and if T cells activate macrophages, their islet toxicity would be linked to recognition of ß cell antigens (9). Inflammatory CD4+ T cells are thought to activate macrophages during host defense against pathogens residing in macrophage endocytic vesicles. Applying this concept of CD4+ T cell-dependent macrophage activation to islet destruction requires presentation of ß cell antigens by MHC class II molecules. In the NOD mouse, ß cells express MHC class I, but not class II, molecules (10), implying that CD4+ T cells can recognize ß cell antigens only indirectly, after shedding of ß cell antigens and subsequent uptake by macrophages.
Primed effector CD8+ T cells can recognize their targets directly and, as such, ß cell destruction by CD8+ T cells would be expected to be independent of accessory cells. On the other hand, any stimulatory cross-talk between ß cell-specific CD8+ T cells and macrophages would establish a link between recognition of antigen presented by ß cells themselves and macrophage activation. Although it is becoming increasingly clear that priming of CD8+ T cells requires antigen presentation by professional antigen presenting cells (11,12), it is less clear to what extent CD8+ T cell effector function is influenced by accessory cells. In previous studies we found that cloned islet-specific as well as islet-derived polyclonal CD8+ T cells are ineffective in destroying islets that are not infiltrated with other cells (13). Similarly, reports by others indicate that it is difficult to demonstrate strong ß cell cytotoxic effector function of islet-derived CD8+ T cells or cloned ß cell-specific CD8+ T cells (1416), although there is convincing evidence that CD8+ T cells as well as MHC class I expression by ß cells are essential for diabetes development (17). In vivo islet inflammation is chronic and does not lead to rapid destruction of ß cells (18). It is possible that islet-infiltrating CD8+ T cells are not strongly activated by ß cells and as a consequence neither rapidly kill nor rapidly undergo activation-induced cell death, but instead persist in the islet infiltrate. In contrast to non-inflamed islets, inflamed islets were effectively destroyed by, and were better activators of, cloned CD8+ T cells. CD8+ T cell-dependent destruction of inflamed islets was prevented by inhibitors of NO synthase, suggesting that among cells in the inflammatory infiltrate, macrophages are the cells that interact with CD8+ T cells in islet-destructive effector function (13). However, direct evidence for the concept that CD8+ T cells can cooperate with macrophages in target destruction was not provided. Moreover, the mechanism by which macrophages cooperate with CD8+ T cells was not studied.
In the present study we have used a bona fide macrophage preparation to provide direct evidence for the notion that CD8+ T cells have the capacity to activate macrophages and to recruit them in target-destructive effector function. Based on our data, we propose a tripartite interaction of ß cells, CD8+ T cells and macrophages during target destruction. CD8+ T cells recognize ß cells antigens directly, but if this leads to only weak activation they may not kill ß cells on their own. However, even weak activation may allow CD8+ T cells to engage in cross-talk with nearby macrophages. This cross-talk does not require presentation of antigen by macrophages to CD8+ T cells. Activated macrophages perform two functions: (i) they co-stimulate CD8+ T cells and (ii) they release NO that facilitates target destruction by CD8+ T cells.
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Methods |
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Reagents and cell culture
Hybridoma YCD3-1 (anti-CD3) was a kind gift from Dr C. A. Janeway, Jr (Yale University, New Haven, CT). TCX6310 cells were kindly provided by Dr F. Melchers (Basel Institute for Immunology, Basel, Switzerland). Antibodies were used in the form of diluted cell culture supernatant or were purified from culture supernatant using GammaBind Plus Sepharose (Pharmacia Amersham, Piscataway, NJ) columns as indicated.
The tissue culture medium (TCM) used for cell culture and all experiments was based on Clicks medium (Irvine Scientific, Santa Ana, CA), which was supplemented with 4 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (Life Technologies), 40 µM 2-mercaptoethanol and 10% FBS (HyClone, Logan, UT). For routine T cell culture we used supernatant of TCX6310 cells (19) as a source of IL-2. For depletion of macrophages, islets were cultured in CMRL 1066 medium supplemented with 1.5 mg/ml glucose, 10% FBS and antibiotics as in TCM.
Cells
To obtain islet-derived CD8+ T cells, islets were prepared from pancreata of 10- to 12-week-old non-diabetic female NOD mice by collagenase digestion (20). Islets were cultured for 23 h in 5 ml TCM. To release infiltrating cells, islets were forced through a 25-gauge needle. The mixture of single cells and pieces of islets was transferred to six-well plates (5 x 105 single cells/well). Mitomycin C-treated spleen cells (5 x 106) and anti-CD3 mAb (culture supernatant of hybridoma YCD3-1 at a final dilution of 1:20) were added to each well. The anti-CD3 mAb was removed after 48 h by washing, and cells were resuspended in TCM supplemented with IL-2 (40 U/ml) and IL-7 (10 ng/ml). After 48 h, cells were fed once more with the same medium. On day 5 after stimulation, islets and clumps of cells were removed using a cell strainer. To remove CD4+ T cells, the cell suspension was treated with anti-CD4 mAb (GK1.5 hybridoma supernatant diluted 1:3, 30 min on ice) followed by pooled rabbit complement diluted 1:10 (45 min, 37°C). Dead cells were removed by centrifugation over lymphocyte separation medium (Organon Teknika, Durham, NC). The resulting cell population contained 91.2 ± 0.9% CD8+ and 1.3 ± 0.3% CD4+ T cells as detected by flow cytometry.
To obtain splenic CD8+ T cells, spleen cells (2 x 106/well, six-well plate) were cultured for 48 h with 3 x 106 mitomycin C-treated spleen cells in 5 ml TCM in the presence of anti-CD3 mAb (culture supernatant of hybridoma YCD3-1 diluted 1:20) for 48 h and then treated as described above. The resulting cell population contained 95.4 ± 1.3% CD8+ and 1.0 ± 0.4% CD4+ T cells. The islet-reactive CD8+ T cell clone 8F7, obtained from the spleen of a newly diabetic NOD female mouse, has been described earlier (13).
Peritoneal exudate cells (PEC) were obtained 4 days after injection of 1 ml of 6% thioglycollate as described (13). PEC were dispersed into culture wells at various dilutions and cultured in TCM for 2 h at 37°C to allow macrophages to adhere to the tissue culture plastic. Non-adherent cells were removed by washing with culture medium. Adherent cells contained >90% of CD11b+ (Mac-1+) cells as determined by flow cytometry.
Assays of islet destruction
Islets were prepared by collagenase digestion as described previously (20). Islets were manually picked using a dissection microscope and cultured overnight in TCM. Five to 10 islets were placed in 96-well flat-bottom plates containing various numbers of macrophages as indicated and 1 x 105 T cells were added. T cells were used 56 days after stimulation. Either T cells or macrophages were omitted as controls. IL-2 was added at 10 U/ml. Islet destruction was determined either by counting the number of remaining islets or morphometrically by determining the islets size. For the counting assay, the number of islets was assessed at regular time intervals by phase contrast microscopy. Details of this assay were described earlier (13). For the more sensitive morphometric assay, electronic images of islets were recorded using video enhanced phase contrast microscopy. The focal plane was set at the equatorial islet perimeter, identified as the largest plane and having a sharp boundary. The electronic images were analyzed further using the NIH Image program (PC version of Scion Image 4ß). The cross-sectional area of islets at the equatorial plane was calculated and used as an estimate of residual islet mass. Unless otherwise indicated, differences were calculated between measurements at 16 and 70 h. In some experiments, destruction of single islets was monitored (experiments in Figs 4B and 6B). They were placed in 96-well round-bottom assay plates together with 5 x 104 CD8+ T cells, 1.5 x 104 macrophages or a combination of both.
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In some experiments, anti-IFN- mAb XMG1.2, anti-LFA-1 (M17/4.4.11.9), anti-ICAM-1 (YN1/1.7.4) mAbs and the NO synthase inhibitor L-NIL were added as indicated in the figure legends. After a 48 h culture, 100 µl of culture medium was collected for measurements of IFN-
and NO. The culture medium withdrawn for sampling was replaced with the same volume of fresh medium containing all supplements.
Assays for IFN- and NO
The concentrations of IFN- in culture supernatants were measured by sandwich ELISA, using paired anti-cytokine antibodies (PharMingen). The sensitivity of the assay was 1 U/ml (67 pg/ml). Levels of NO released into culture medium were measured using Griess reagent (21). Solutions of NaNO2 were used as standards. The sensitivity of the assay was 0.2 µM NO2.
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Results |
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To directly test whether CD8+ T cells cooperate with macrophages during islet destruction, islets from NOD-scid mice (a model for non-inflamed islets) were cultured with CD8+ T cells expanded from inflamed NOD islets in the presence or absence of bona fide macrophages, which were prepared from peritoneal exudate. Early stages of islet destruction are shown in Fig. 1(A). In the absence of macrophages, islet-derived CD8+ T cells were ineffective in islet destruction, as were added macrophages without T cells. In contrast, co-culture of CD8+ T cells, macrophages and islets resulted in islet destruction. A morphometric assay, used to quantitate the decrease of islet area during islet destruction, confirmed that islet-derived CD8+ T cells effectively cooperated with macrophages during islet destruction (Fig. 1B). In contrast, CD8+ T cells expanded from the spleen of NOD mice were much less effective (Fig. 1B), suggesting that the islet infiltrate contains an increased frequency of ß cell-specific CD8+ T cells. Consistent with this notion, cloned ß cell-specific CD8+ T cells [clone 8F7 (13)] were even more effective than either islet- or spleen-derived CD8+ T cells in islet destruction (Fig. 1C). Increasing numbers of macrophages both enhanced and accelerated islet destruction by cloned CD8+ T cells (Fig. 1C). Macrophages without CD8+ T cells, even at the highest dose, did not destroy islets. These results clearly demonstrate that both islet-derived and cloned CD8+ T cells cooperate effectively with macrophages during islet destruction.
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Cooperative islet destruction requires antigen presentation by ß cells, but not by macrophages
Among contact-dependent events, antigen presentation at the ß cellCD8+ T cell interface, at the macrophageCD8+ T cell interface, or both, may be required for cooperative islet destruction. To address this question, MHC-mismatched macrophages were examined. CD8+ T cell clone 8F7 is H-2Db restricted and not alloreactive to H-2Dd (13). As shown in Fig. 4(A), the macrophageCD8+ T cell cooperative effect could be reproduced with MHC-mismatched BALB/c macrophages (BALB/c:H-2d versus NOD:H-2Kd, H-2Db), although BALB/c macrophages were less effective than NOD macrophages.
To confirm these observations and to test a requirement for antigen presentation by ß cells, antigen presentation was eliminated from either ß cells or macrophages and islet destruction was examined. Macrophages from ß2m-deficient NOD mice, which cannot present antigen, were as effective as macrophages from wild-type NOD mice (Fig. 4B). In contrast, islets from ß2m-deficient mice, which contain ß cells that cannot present antigen, were not destroyed, even in the presence of macrophages from wild-type mice (Fig. 4B). These data suggest a requirement for antigen presentation by ß cells to CD8+ T cells; however, the interactions between CD8+ T cells and exogenously added macrophages are not dependent on the ability of macrophages to present antigen.
CD8+ T cells and macrophages cross-stimulate each other
Contact-dependent and ICAM-1/LFA-1-mediated interactions could play a role not only at the CD8+ T cellß cell interface, but also at the CD8+ T cellmacrophage interface. Indeed, in the transwell experiment shown in Fig. 3(A) some islet destruction occurred when CD8+ T cells were co-localized on one side of the porous membrane and islets on the other, but not when CD8+ T cells and macrophages were separated. To test if CD8+ T cells and macrophages can cross-stimulate each other, samples from the transwell experiment shown in Fig. 3 were analyzed for IFN- secretion and NO release as markers of CD8+ T cell and macrophage activation respectively. CD8+ T cells and macrophages potently stimulated each other when they were co-localized (Fig. 5A). Similar results were obtained when pre-activated CD8+ T cells and macrophages were co-cultured without islets. Antibodies specific for either ICAM-1 or LFA-1 blocked both IFN-
secretion and NO release (Fig. 5B and C), while isotype-matched control antibodies had no effect (not shown). These data demonstrate that CD8+ T cells and macrophages cross-stimulate each other to produce IFN-
and NO. The data in Figs 3(A and B) and 5(A) taken together suggest that the production of soluble factors acting at a distance can cause some damage, but is not sufficient for complete islet destruction.
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These data suggest that CD8+ T cells and macrophages cross-stimulate each other and that signals derived from CD8+ T cells activate macrophages to produce NO which plays a significant role in accelerated tissue destruction in this system.
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Discussion |
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Several previous reports have suggested a role of macrophages in the activation of CD8+ T cells either by presenting antigen or by providing co-stimulatory activity (11,12,14,25). In a previous paper we have provided evidence that implicates macrophages in CD8+ T cell effector function (13). The present paper provides more direct evidence for the concept that CD8+ T cells can cooperate with macrophages in target destruction and characterizes the mechanism by which this cooperation can occur. In contrast to the previous study, most of the experiments in this study were performed with macrophages from the peritoneal exudate rather than with inflamed islets, presumed to contain endogenous macrophages. All evidence available to us so far suggests that both populations of macrophages interact with CD8+ T cells in similar ways. Both co-stimulate CD8+ T cells as determined by IFN- release and, for both, blocking the inducible form of NO synthase inhibits islet destruction.
Although the present study was performed with islets of Langerhans and CD8+ T cells from the NOD mouse, it remains to be investigated how important the proposed mechanism is in vivo during the development of type 1 diabetes and whether or not it also applies to other types of target destruction by CD8+ T cells, such as tumor rejection, transplant rejection, demyelination during the development of multiple sclerosis and experimental autoimmune encaphalomyelitis, and host defense against chronic infection with viruses. It is possible that the ability of NOD CD8+ T cells and macrophages to potently cross-stimulate each other is an abnormality of the NOD mouse.
Like many other tissues, islets are assemblies of tightly adherent cells and their destruction is likely to be more complex than target destruction in more classical cytolytic T cell assays, typically employing dispersed single cells. The present data clearly show that CD8+ T cells cooperate with macrophages during islet destruction; however, the question as to the ultimate lethal insult is not addressed. Soluble macrophage products such as NO do not appear to be sufficient for complete islet destruction, whereas TCR engagement is essential. It is therefore likely that macrophages and macrophage products play a facilitating role, but target destruction is completed by effector mechanisms of CD8+ T cells, such as perforin or FAS ligand. Preliminary data show that the integrity of islet tissue is diminished by NO, suggesting that one of NOs functions may be to facilitate access of CD8+ T cells to ß cells.
The data show that contact is necessary for complete islet destruction. However, cooperating CD8+ T cells and macrophages were able to inflict some initial damage to islets even without contact, suggesting that for this to occur recognition of ß cells by CD8+ T cells is not an absolute requirement. Pre-activated CD8+ T cells may have a diminished requirement for the signal provided by recognition of ß cell antigen. The data in the T cellmacrophage cross-stimulation experiment (Fig. 5B and C) suggest that ICAM-1/LFA-1 interactions may play a role not only in the interaction of ß cells with CD8+ T cells (26), but also in the antigen-independent cross-stimulation of CD8+ T cells and macrophages. Upon activation of T cells, the avidity of LFA-1 for ICAMs increases and ICAM-1 expression is up-regulated in activated macrophages. Both events may facilitate the cross-talk between CD8+ T cells and macrophages. In addition to merely mediating contact, the ligation of ICAM-1 by LFA-1 may provide an activating signal to macrophages. A role of ICAM-1 in the activation of Kupffer cells has been suggested (27), but a role of LFA-1/ICAM-1 interaction in CD8+ T cell-dependent macrophage activation has, to our knowledge, not been previously reported. The observations that (i) pre-activated CD8+ T cells and macrophages can cross-stimulate each other without continued engagement of the TCR, and (ii) that some islet damage can occur when islets are separated from CD8+ T cells and macrophages, suggests that in vivo some islet-damage may even occur if, for whatever reason, non-specific, but activated, CD8+ T cells enter islets that already contain macrophages, such as islets of young NOD mice (28).
The soluble factors IFN- and NO play a role in cooperative islet destruction, IFN-
being less important than NO. It is controversial whether or not IFN-
or NO are important for islet destruction and disease development in vivo. IFN-
knockout mice still develop disease, although blocking IFN-
signaling post-natally does impair disease development (29). These seemingly contradictory findings may be explained by the complex role of IFN-
on T cell development and regulation. If absent from birth, alternative mechanisms may compensate for the role of IFN-
. Similar arguments can be made for the role of NO in diabetes development. Unpublished data appear to suggest that the inducible form of NO synthase is not essential for diabetes development in the NOD mouse (30). NO has important regulatory functions at many points of a developing immune response (30) which may obscure any facilitating role of CD8+ T cell-dependent islet destruction.
The data in this report demonstrate that CD8+ T cells from the islet infiltrate do not readily destroy islets or ß cells in the absence of macrophages. Weak killing activity by islet-derived CD8+ T cells has been reported and reviewed by others (1416). In contrast, inflamed islets, containing a large number of macrophages, were readily destroyed (13). The present data offer a potential explanation for these earlier findings. Even if CD8+ T cells are only weakly activated by target cells and cannot kill them on their own, they may still be able cross-talk with inflammatory macrophages which facilitates target destruction.
The weak activation of cytotoxic T lymphocyte effector function may paradoxically facilitate chronic persistence of CD8+ T cells and long-term damage of islets. The activationinactivation cycle of pathogenic CD8+ T cells in inflammatory conditions such as type 1 diabetes may be different from that of CD8+ T cells that clear infections. During host defense against acute infection, the T cell response is typically vigorous, but short lived, leading to the clearance of the infection. During the down-regulation of a pathogen-specific immune response most antigen-specific CD8+ T cells undergo apoptosis and some develop into memory cells. Most effector CD8+ T cells have a very limited life span. In contrast, the presence of T cells in the islet inflammatory infiltrate is chronic and the autoimmune response is long-lived. One interpretation that is consistent with the chronicity of the infiltrating CD8+ T cell pool is that CD8+ T cells in the islet are too weakly activated to either kill ß cells on their own or to rapidly undergo activation-induced apoptosis. We propose that even if this activation is too weak for killing, CD8+ T cells can still cross-talk with macrophages. This may facilitate slow cooperative islet destruction.
Even if CD8+ T cells are activated, low levels of NO released by macrophages could protect T cells from activation-induced apoptosis (30). Compared to ß cells (31), CD8+ T cells are not very sensitive to the toxic effects of higher concentrations of NO (32). In our system, only islets are destroyed; CD8+ T cells and macrophages survive. NO is probably not the only protective factor. We and others have shown that inflamed NOD islets produce large amounts of prostaglandin E2 (33). Although it inhibits the killing function of CD8+ T cells, it also inhibits activation-induced apoptosis (34).
The interaction of CD8+ T cells is a potentially deadly encounter for macrophages. CD8+ T cells would possibly kill macrophages if ß cell antigens were presented to CD8+ T cells after they have developed effector function (35). However, because the CD8+ T cellmacrophage interaction is not based on presentation of ß cell antigen to CD8+ T cells, and may not occur initially, MHC-restricted cytotoxic T lymphocyte activity is not directed towards macrophages. Furthermore, NOD macrophages aberrantly down-regulate expression of MHC class I molecules in response to IFN- (36) which could contribute to their protection from CD8+ T cells.
The cooperation of CD8+ T cells and macrophages during islet destruction proposed in this paper does not exclude an equally important role for CD4+ T cells. Indeed, our unpublished data show that CD4+ T cells also can trigger NO production in NOD macrophages.
The dependence on macrophages varies for different clonal lines of CD8+ T cells. Although the insulin-specific clone C9G8 (15,37) also cooperates with macrophages, it is less dependent on them than clone 8F7 or polyclonal CD8+ T cells from the islet infiltrate. A summary scheme accounting for varying degrees of macrophage dependence is shown in Fig. 7. If the peptideMHC combination that interacts with the CD8+ T cell is a strong agonist, macrophages may be dispensable. Presumably, this is the case for very few peptides that exhibit perfect structural and chemical complementarity to the TCR. In this case the recognition of the target is highly specific. If, on the other hand, the MHC-bound peptide exhibits only imperfect chemical and structural complementarity to the TCR, a requirement that can be met by more peptides, the requirement for cooperation with macrophages increases and target destruction becomes less specific. At the extreme end of this spectrum, the CD8+ T cells do not recognize the target at all, but if they are pre-activated and can exchange cross-activating signals with macrophages, some non-specific target destruction may still occur.
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Acknowledgements |
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Abbreviations
L-NILL-N6-(1-iminoethyl)lysineHCl
Mmacrophage
NODnon-obese diabetic
PECperitoneal exudate cell
TCMtissue culture medium
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References |
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