Antigen-independent cross-talk between macrophages and CD8+ T cells facilitates their cooperation during target destruction

Tatyana Gurlo1 and Hermann von Grafenstein1,2

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


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Inflammatory sites associated with tissue destruction often contain a complex mixture of cells including macrophages as well as CD8+ and CD4+ T cells. Here, we have investigated, using islets of Langerhans as targets, if CD8+ T cells and macrophages can cooperate in tissue destruction. CD8+ T cells obtained from the islet inflammatory lesion of non-obese diabetic mice or cloned islet-specific CD8+ T cells were ineffective in destroying islets on their own. Including increasing numbers of macrophages in co-cultures of islets and islet-derived or cloned CD8+ T cells progressively increased and accelerated islet destruction. Macrophages alone were ineffective. Macrophage-depleted islets were not destroyed by islet-derived CD8+ T cells. For cooperative islet destruction to occur, ß cells, but not macrophages, needed to be able to present antigens to CD8+ T cells. CD8+ T cells triggered NO production by macrophages, while macrophages triggered IFN-{gamma} production by CD8+ T cells. Each of these factors was partially effective, but not sufficient, for maximal islet destruction. Antibodies specific for ICAM-1 and LFA-1 inhibited both cooperative islet destruction and cross-stimulation of CD8+ T cells and macrophages. The data suggest that if CD8+ T cells become only weakly activated by target cells, they are not able to destroy target tissue on their own. However, such CD8+ T cells and local macrophages may still cross-stimulate each other, which then facilitates target destruction. For this to occur, target cells, but not macrophages, need to present antigen to CD8+ T cells.

Keywords: cell–cell interaction, ICAM-1, LFA-1, NO, type 1 diabetes


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The islets of Langerhans of the non-obese diabetic (NOD) mouse contain chronic inflammatory infiltrates that lead to eventual destruction of insulin-producing ß cells (1,2). Islet inflammation and ß cell destruction are also hallmarks of human type 1 diabetes and the NOD mouse has therefore been extensively studied as a model for this disease. Similar to many other inflammatory lesions associated with tissue destruction, the islet inflammatory infiltrate contains a complex mix of cells including macrophages, dendritic cells and B cells, as well as CD8+ and CD4+ T cells. Although each of these cell types have been shown to be essential for disease development (37), the interplay of different cell types in the inflammatory infiltrate is incompletely understood. Using islets of Langerhans as a model target, we have focused in this study on cross-talk between CD8+ T cells and macrophages, and the potential role of this cross-talk in cooperative target destruction.

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.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
NOD/Lt, NOD-scid, ß2m–/–-NOD-scid and BALB/c mice were obtained from the Jackson Laboratory (Bar Harbor, ME), and maintained or bred in the University of Southern California Animal Facility under pathogen-free conditions. The spontaneous incidence of diabetes in our NOD colony reaches 65–70% in female mice by 20 weeks of age and diabetes usually commences by 13 weeks of age. For some experiments NOD female mice were obtained from Taconic Farm (Taconic, Germantown, NY). Mice aged 8–12 weeks were used for experiments.

Reagents and cell culture
Hybridoma YCD3-1 (anti-CD3{epsilon}) 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 Click’s 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 2–3 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{epsilon} 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{epsilon} 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 5–6 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 islet’s 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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Fig. 4. CD8+ T cell–macrophage cooperation during islet destruction requires antigen presentation by ß cells, but not by macrophages. (A) Cloned CD8+ T cells cooperate with MHC-mismatched macrophages. NOD-scid islets were cultured with 1 x 105 cloned 8F7 CD8+ T cells (Db restricted) in the presence of the indicated number of BALB/c (H-2d) macrophages. Data are the mean ± SEM of three experiments. Islet destruction was assessed as in Fig. 1C. (B) Single islets from NOD-scid or ß2m–/–-NOD-scid (NODscidB2m) mice were cultured with 5 x 104 CD8+ T cells derived from NOD islets and 1.5 x 104 macrophages from NOD-scid or ß2m–/–-NOD-scid (NODscidB2m) mice. Data are the mean ± SEM of three experiments. Islet destruction was assessed morphometrically.

 


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Fig. 6. Role of IFN-{gamma} and NO in macrophage–CD8+ T cell cooperative islet destruction. NOD-scid islets were cultured with 1 x 105 islet CD8+ T cells in the presence of 5 x 104 thioglycollate-induced peritoneal macrophages from NOD mice. A neutralizing anti-IFN-{gamma} mAb (A) or L-NIL, a specific inhibitor of the inducible form of NO synthase (B) was added as indicated. Islet destruction was monitored morphometrically. Data are the mean ± SEM of five measurements from one of two similar experiments.

 
To study the influence of co-localization, islets, CD8+ T cells and macrophages were placed either on the same or opposite sides of 0.4-µm pore size transwell filters (Corning Costar, Cambridge, MD).

In some experiments, anti-IFN-{gamma} 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-{gamma} and NO. The culture medium withdrawn for sampling was replaced with the same volume of fresh medium containing all supplements.

Assays for IFN-{gamma} and NO
The concentrations of IFN-{gamma} 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.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD8+ T cells cooperate with macrophages during islet destruction
Previous experiments have shown that inflamed islets are better targets than non-inflamed islets for CD8+ T cell-mediated destruction (13). These experiments implicated macrophages in the enhancing effect of inflammation because an antagonist of the inducible form of NO synthase inhibited this effect.

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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Fig. 1. CD8+ T cells cooperate with exogenous macrophages during islet destruction. Non-inflamed (NOD-scid) islets were cultured with various CD8+ T cells as indicated, and thioglycollate-induced peritoneal NOD macrophages (M{phi}). (A) Phase contrast micrographs of co-cultures of islets and various combinations of islet-derived CD8+ T cells (1 x 105) and macrophages (5 x 104). Magnification ~x100. (B) Morphometric quantitation of islet destruction by measuring the decrease of islet area following incubation with various numbers of macrophages without T cells (crosses), or with macrophages and 1 x 105 CD8+ T cells derived from islets (circles) or spleen (triangles). (C) Islet destruction by various numbers of macrophages as indicated and 1 x 105 cloned (8F7) CD8+ T cells. Islets remaining at the indicated time intervals after start of the co-culture were visualized by phase contrast microscopy and counted. Data are the mean ± SEM of three experiments.

 
Islets which exhibit no obvious inflammation contain a small number of endogenous macrophages (22). If the above hypothesis is correct, it would be expected that depleting endogenous macrophages would diminish the minimal islet-destructive effect observed without exogenously added macrophages. To deplete endogenous islet macrophages, islets from NOD-scid mice were cultured for five days at reduced temperature (22,23). Consistent with our hypothesis, macrophage-depleted islets were more resistant to destruction by islet-derived CD8+ T cells compared with macrophage-containing islets (Fig. 2). This result shows furthermore that endogenous macrophages cooperate, similarly to exogenously added peritoneal macrophages, with CD8+ T cells in islet destruction.



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Fig. 2. CD8+ T cells cooperate with endogenous islet macrophages. NOD-scid islets (control) or NOD-scid islets depleted of endogenous macrophages were cultured with 1 x 105 islet-derived CD8+ T cells. Destruction was monitored morphometrically and data are the mean ± SEM of 33 measurements from two separate experiments.

 
Islet-destructive CD8+ T cell–macrophage cooperation is contact dependent, and mediated by LFA-1 and ICAM-1
Cooperation between CD8+ T cells and macrophages during islet destruction could be contact dependent or independent. Contact independence could be mediated via release of soluble factors. These factors could act at a distance or require close proximity to be effective. To examine the requirement for close proximity, CD8+ T cells, macrophages and islets were placed either on the same or opposite sides of transwell chambers. Islet destruction was maximal and proceeded to completion only when CD8+ T cells, macrophages and islets where localized on the same side of the porous membrane (Fig. 3). CD8+ T cells alone were ineffective in islet destruction and macrophages co-localized with islets, but separated from CD8+ T cells, did not destroy islets (Fig. 3). Some islet destruction occurred when islet CD8+ T cells were placed together with macrophages and islets on the other side (Fig. 3A), suggesting that interaction between macrophages and CD8+ T cells results in release of soluble factors. However, under these suboptimal conditions, islet destruction was limited and did not proceed to completion (Fig. 3B). To ensure that the results are a reflection of co-localization and not an artifact of different culture conditions in the inner versus the outer chamber, the distribution of the cells in the transwell chambers was reversed. This did not change the results, indicating that they depended on co-localization, not location in the transwell insert or outer chamber. These data indicate that all three cell types need to be in close proximity for a maximal islet destructive effect.



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Fig. 3. Proximity of CD8+ T cells and macrophages is required for islet destruction. NOD-scid islets (Isl.) were cultured with 8F7 cloned CD8+ T cells (1 x 105) and NOD macrophages (2.5 x 104) in transwell chambers. The line (A and B) indicates how cells were placed on one or the other side of the porous membrane (0.4 µM pore size). (A) The change in islet size over 40 h was determined morphometrically. (B) Islet destruction was assessed by phase contrast microscopy after 48 h. The number of islets lost (no residual islet mass detected) is plotted. Data are the mean ± SEM of measurements of 10 islets from one of three similar experiments. (C) Role of LFA-1 and ICAM-1. NOD-scid islets were cultured with 1 x 105 islet CD8+ T cells and 5 x 104 NOD macrophages in the presence of 10 µg/ml anti-LFA-1 ({alpha}-LFA-1), anti-ICAM-1 ({alpha}-ICAM-1) mAb or isotype-matched IgG (IgG2a). Islet destruction was monitored morphometrically. Data are the mean ± SEM of five measurements from one experiment of three performed.

 
To probe contact dependence of cooperative islet destruction by CD8+ T cell and macrophages, and to examine which molecules mediate such contact-dependent interactions, a requirement for adhesion molecules was addressed. Activated T cells express LFA-1 which may bind to ICAM-1 expressed by macrophages or ß cells. Figure 3(C) shows that a neutralizing anti-LFA-1 mAb strongly inhibited the islet-destructive cooperative effect. A neutralizing anti-ICAM-1 mAb was also inhibitory (Fig. 3C), although somewhat less effective. Isotype-matched control antibodies had no effect alone or when included with the LFA-1/ICAM-1 antibodies. The data suggest that LFA-1/ICAM-1-mediated contact plays a key role in cooperative islet destruction.

Cooperative islet destruction requires antigen presentation by ß cells, but not by macrophages
Among contact-dependent events, antigen presentation at the ß cell–CD8+ T cell interface, at the macrophage–CD8+ 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 macrophage–CD8+ 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 cell–ß cell interface, but also at the CD8+ T cell–macrophage 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-{gamma} 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-{gamma} 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-{gamma} 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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Fig. 5. LFA-1- and ICAM-1-dependent cross-talk between CD8+ T cells and macrophages. (A) Levels of NO and IFN-{gamma} production were measured in the supernatant collected from the transwell experiment shown in Fig. 3(A). The data are normalized relative to release that occurred when islets, macrophage and CD8+ T cells were on the same side of the transwell membrane. (B) NO synthesis by macrophages triggered by CD8+ T cells. (C) IFN-{gamma} synthesis by TCR pre-stimulated CD8+ T cells triggered by macrophages. (B and C) Islet-derived CD8+ T cells (1 x 105) were cultured with NOD macrophages (5 x 104) in the presence of the indicated concentrations of antibodies ({alpha}-LFA-1 and {alpha}-ICAM-1). After 48 h, samples were withdrawn from the supernatant for measurement of NO and IFN-{gamma}. Data are the mean ± SEM of four measurements from two experiments (B) and the mean of two measurements from one experiment of two performed (C). Background NO production in the absence of CD8+ T cells was undetectable and background IFN-{gamma} release was <10% of macrophage-triggered IFN-{gamma} release.

 
Even though NO and IFN-{gamma} production are not sufficient for optimal cooperative islet destruction, they may still be necessary. To address this question, an antibody specific for IFN-{gamma} was used to neutralize IFN-{gamma} in the CD8+ T cell–macrophage co-culture system described above and an inhibitor of the inducible form of NO synthase (L-NIL) was used to block NO synthesis. Cooperative islet destruction was indeed inhibited by a neutralizing anti-IFN-{gamma} mAb, although this effect was only partial (Fig. 6A). Control experiments confirmed that all of the IFN-{gamma} was neutralized by the antibody. L-NIL inhibited macrophage-aided islet destruction triggered by both islet-derived CD8+ T cells (Fig. 6B) and cloned 8F7 CD8+ T cells (data not shown). These results do not formally exclude ß cells as a potential source of NO, but still implicate macrophages because ß cell NO synthesis depends on the macrophage product IL-1 (24).

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.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The data in this report suggest a new paradigm for CD8+ T cell-dependent target destruction, i.e. CD8+ T cells cooperate with macrophages based on LFA-1- and ICAM-1-dependent cross-talk between both cell types. Cooperative target destruction requires recognition of target cell antigens by CD8+ T cells; however, macrophages do not need to present antigens to CD8+ T cells.

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-{gamma} 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 NO’s 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 cell–macrophage 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-{gamma} and NO play a role in cooperative islet destruction, IFN-{gamma} being less important than NO. It is controversial whether or not IFN-{gamma} or NO are important for islet destruction and disease development in vivo. IFN-{gamma} knockout mice still develop disease, although blocking IFN-{gamma} signaling post-natally does impair disease development (29). These seemingly contradictory findings may be explained by the complex role of IFN-{gamma} on T cell development and regulation. If absent from birth, alternative mechanisms may compensate for the role of IFN-{gamma}. 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 activation–inactivation 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 cell–macrophage 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-{gamma} (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 peptide–MHC 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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Fig. 7. A model of CD8+ T cell–macrophage cooperation. CD8+ T cells that are strongly activated upon recognition of antigen presented by target cells can destroy their target independently of macrophages. This is highly specific target destruction. At intermediate levels of activation through their TCR, CD8+ T cells become more and more dependent on macrophages. Bi-directional cross-talk with macrophages mediated by LFA-1 and ICAM-1 facilitates target destruction, but the specificity of target destruction decreases. If CD8+ T cells do not recognize their target at all, but have recently been activated, they may undergo signal exchange with macrophages. This may lead to non-specific target destruction.

 
The data in this report confirm and extend earlier data (13,14) suggesting that macrophages co-stimulate CD8+ T cells. The cross-talk between CD8+ T cells and macrophages is therefore reciprocal, and may constitute a positive feed-forward loop. Although this loop may also occur in strains of mice that do not develop autoimmunity, the strength of the coupling between CD8+ T cells and macrophages may determine whether this signaling loop is either self-enhancing and unstable (38), leading to autoimmunity, or abortive, as in healthy strains of mice.


    Acknowledgements
 
Supported by National Institutes of Health grant DK49717 to H. v. G. We thank Dr Peter Butler, University of Southern California, for support and helpful discussions during the course of this work, and Drs Steven Stohlman and Cornelia Bergmann, University of Southern California, for critically reading this manuscript.

Abbreviations
L-NIL—L-N6-(1-iminoethyl)lysine–HCl

M{phi}—macrophage

NOD—non-obese diabetic

PEC—peritoneal exudate cell

TCM—tissue culture medium


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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