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The Journal of Immunology, 2003, 171: 2216-2224.
Copyright © 2003 by The American Association of Immunologists

Microglia-Mediated Nitric Oxide Cytotoxicity of T Cells Following Amyloid {beta}-Peptide Presentation to Th1 Cells 1

Alon Monsonego2, Jaime Imitola, Victor Zota, Takatoku Oida and Howard L. Weiner

Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Alzheimer’s disease is marked by progressive accumulation of amyloid {beta}-peptide (A{beta}) which appears to trigger neurotoxic and inflammatory cascades. Substantial activation of microglia as part of a local innate immune response is prominent at sites of A{beta} plaques in the CNS. However, the role of activated microglia as A{beta} APCs and the induction of adaptive immune responses has not been investigated. We have used primary microglial cultures to characterize A{beta}-Ag presentation and interaction with A{beta}-specific T cells. We found that IFN-{gamma}-treated microglia serve as efficient A{beta} APCs of both A{beta}1–40 and A{beta}1–42, mediating CD86-dependent proliferation of A{beta}-reactive T cells. When cultured with Th1 and Th2 subsets of A{beta}-reactive T cells, Th1, but not Th2, cells, underwent apoptosis after stimulation, which was accompanied by increased levels of IFN-{gamma}, NO, and caspase-3. T cell apoptosis was prevented in the presence of an inducible NO synthase type 2 inhibitor. Microglia-mediated proliferation of A{beta}-reactive Th2 cells was associated with expression of the Th2 cytokines IL-4 and IL-10, which counterbalanced the toxic levels of NO induced by A{beta}. Our results demonstrate NO-dependent apoptosis of T cells by A{beta}-stimulated microglia which may enhance CNS innate immune responses and neurotoxicity in Alzheimer’s disease. Secretion of NO by stimulated microglia may underlie a more general pathway of T cell death in the CNS seen in neurodegenerative diseases. Furthermore, Th2 type T cell responses may have a beneficial effect on this process by down-regulation of NO and the proinflammatory environment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The brain has evolved to limit access of the immune system. This limitation is mediated by mechanisms such as the physical blood-brain barrier and a molecular milieu that suppresses immune function (1, 2, 3). The CNS is thus often described as "immune privileged." However, a body of evidence suggests that T cells normally, although to a limited extent, survey the CNS (4, 5, 6, 7, 8). This ability of T cells to cross the blood-brain barrier has primarily been described for activated cells that survey the organs of the body during an immune response. Death of T cells in the CNS, however, has been characterized as very intense, specifically under inflammatory conditions (9), and hence the migration of T cells out of the CNS, unlike their migration from peripheral tissues, has not been reported. It has been suggested that T cell death occurs primarily via activation-induced cell death with Fas/Fas ligand (FasL) 3 interactions playing a major role (2, 10), since FasL is highly expressed in the CNS by astrocytes, microglia, and neurons.

To elicit T cell activation in the CNS, an Ag must be phagocytized and processed by local APCs. The professional APCs of the CNS are the parenchymal microglia and the perivascular cells (11, 12, 13). The parenchymal microglia have recently been characterized as myeloid progenitors cells that can differentiate into macrophage-like or dendritic-like cells (14, 15, 16, 17).

Differentiation of microglia and its effect on T cell activation may relate to brain pathology, such as that induced by amyloid {beta}- peptide (A{beta}) in Alzheimer’s disease (AD). A{beta} is a cleavage product of neuronal amyloid precursor protein (18). Cleavage can yield either A{beta}1–40 or A{beta}1–42, and their accumulation and aggregation in cognitive brain regions during aging is a hallmark of AD pathology (19). A{beta}1–42 is the more aggregated form and is more highly correlated than A{beta}1–40 with disease and neurotoxicity. Extensive studies have demonstrated that these A{beta} plaques are colocalized with activated microglia and astrocytes, implicating additional neurotoxicity (20). These pathways include the activation of complement, secretion of the proinflammatory cytokines IL-1{beta}, TNF-{alpha}, and IL-6, and the secretion of NO (21, 22, 23, 24, 25). A{beta} or its fibrillated form may, therefore, be recognized as an Ag that needs to be cleared and provokes activation of microglia and astrocytes. If their activation fails to clear the Ag, this response becomes chronic and neurotoxic.

Microglia, however, have also been found to play a protective role in mouse models of AD. A recent study has shown that overexpression of TGF-{beta} in the brains of these animals significantly decreases A{beta} burdens in the CNS, suggesting that altered activation of the microglial cells may contribute to that effect (26). Immunization with A{beta} also reduced deposition of A{beta} in a mouse model of AD. In this approach, Abs to A{beta} were found to play a significant role in the clearance of A{beta}, suggesting uptake of A{beta} by microglia through the Fc receptors (27). When A{beta} was administered to animals through the nasal route, they cleared A{beta} (28). The clearance was associated with Ab production and the detection of Th2-type cytokines, suggesting a possible beneficial role for infiltrating regulatory T cells that would require activation of microglia to APCs.

In AD, microglia undergo chronic activation as part of the innate immune response and contribute to neurotoxicity. Nevertheless, T cells are rarely observed at sites of A{beta} plaques. In this study, we used primary cultures of microglia to characterize the interaction of microglia with Th1- and Th2-type T cells following stimulation with A{beta}.


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

A{beta}1–40 and A{beta}1–42 synthetic peptides were synthesized in the Biopolymer Laboratory, Center for Neurologic Diseases, Brigham and Women’s Hospital (Boston, MA) or purchased from BioSource International (Camarillo, CA). A{beta} peptides used for T cell assays were dissolved in DMSO (2 mg/ml).

Preparation of cultures of mouse brain microglia

Glial cultures were prepared as follows: cells were dissociated from the cerebral cortex of 1-day-old C57BL/6 mice, carefully removing meninges tissue, and were cultured in poly-D-lysine-coated tissue culture flasks (two brains per 85-cm2 flask) in medium supplemented with DMEM, 4 mM L-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 10 mM nonessential amino acids, 57.2 mM 2-ME (Sigma-Aldrich, St. Louis, MO), and 10% FCS. The medium was changed after 24 h and every 2 days thereafter. On day 7, the culture was incubated with 100 pg/ml IFN-{gamma} for 72 h and, on day 10, the entire glial culture was trypsinized and microglia were labeled with FITC-conjugated anti-CD11b and sorted using a FACSVantage SE Cell Sorter (BD Biosciences, Franklin Lakes, NJ).

Generation of T cell lines

C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) were immunized by footpad injection. Each mouse received 100 µl of human A{beta}1–40 (1 µg/µl; synthesized in the Biopolymer Laboratory, Center for Neurologic Diseases, Brigham and Women’s Hospital), emulsified in an equal volume of CFA containing 50 µg of Mycobacterium tuberculosis. Ten days later, popliteal draining lymph nodes were excised from the mice. Popliteal lymph node-derived lymphocytes were washed in HBSS and then cultured in 24-well plates in DMEM, supplemented as described for the microglia culture, in the presence of 20 µg/ml human A{beta}1–40. On day 10, cells were restimulated with irradiated splenocytes and tested for Ag-induced proliferation and cytokine production. For cytokine measurements by ELISA, supernatants were collected at 24 h for IL-2 and IL-4 and at 40 h for IL-10 and IFN-{gamma}. For proliferation measurements, cells were pulsed with 1 µCi [3H]thymidine/well 48 h after stimulation and harvested 12 h later and then assayed for [3H]thymidine incorporation. T cell lines were maintained by stimulating the cells every 14 days with irradiated splenocytes and 20 µg/ml A{beta}1–40. Half of the medium was replaced with fresh medium supplemented with 10 U/ml IL-2 two days after T cell stimulation and every 2 days thereafter.

T cell stimulation by microglia

Microglia were cultured in flat-bottom 96-well plates in the DMEM-FCS medium after their removal from the glial culture. Two hours later, A{beta}-reactive T cells were added to the culture along with synthetic human A{beta}1–40 or A{beta}1–42 (BioSource International) (see figure legends for cell counts and A{beta} concentrations). Cytokines were measured by ELISA and T cell proliferation was performed as described for T cell lines. Blocking experiments were performed in the presence of hamster anti-mouse CD80, rat anti-mouse CD86, rat anti-mouse IFN-{gamma}, rat anti-mouse IL-10, rat anti-mouse FAS-L (R&D Systems, Minneapolis, MN), CTLA4-Ig (gift from Dr. R. Peach, Princeton, NJ), hamster anti-CD40-L (MR1; Bioexpress Cell Culture Services, West Lebanon, NH), and L-N6-(1-iminoethyl)-lysine · 2 HCl (L-NIL; Alexis Biochemical, San Diego, CA). All Abs were purchased from BD PharMingen (San Diego, CA) if not mentioned otherwise.

Flow cytometry

Microglia cells were stained in U-bottom 96-well plates (Costar, NewYork, NY). Cells were stained for cell surface molecules either directly with FITC- or PE-conjugated Abs or with biotinylated Abs followed by APC-labeled streptavidin. Staining was performed in PBS containing 2% FCS for 20 min on ice followed by three washes. The following Abs (BD PharMingen) were used according to the manufacturer’s instructions: rat anti-mouse CD11b, rat anti-mouse CD4, and isotype controls (see figure legends for specific fluorescence). For intracellular staining, cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) for 20 min. After cells were washed, they were resuspended in permeability buffer (BD PharMingen) and stained with PE-conjugated rabbit anti-caspase-3 (BD PharMingen).

ELISA

Ag-induced cytokine production was measured by sandwich ELISA as described previously (29). Recommended pairs of Abs (coating and detecting) for IL-2, IL-4, IL-10, and IFN-{gamma} were purchased from BD PharMingen and used according to the manufacturer’s instructions. Cytokines also were measured by Pierce, Boston Technology Center (Woburn, MA).

Immunocytochemistry

For immunocytochemical analysis of microglia, cells were cultured on tissue culture glass slides (BD Biosciences; Franklin Lakes, NJ) and fixed with ice-cold methanol:acetone (1:1) for 10 min at -20°C. Cells were washed and incubated for 1 h at room temperature with one of the following Abs: FITC-labeled mouse anti-inducible NO synthase (iNOS), PE-labeled rat anti-mouse IL-10, or their isotype controls (BD Biosciences). Cells were washed and covered with mounting medium and then examined under a Zeiss Laser Scanning Confocal Microscope and three-dimensional analysis software (Zeiss, Thornwood, NY). Washing solutions contained PBS and 0.05% Tween 20. Diluting solutions consisted of PBS containing 3% normal goat serum and 2% BSA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IFN-{gamma}-treated microglia are efficient A{beta} APCs and support CD86-dependent T cell activation

To determine whether microglia can serve as APCs for the stimulation of A{beta}-reactive T cells, we prepared primary glial cultures from the cortex of 1-day-old C57BL/6 mice. At day 7, IFN-{gamma} was added to the medium; at day 10, cells were trypsinized to detach them from the flask and stained with FITC-labeled CD11b Abs. CD11b+ microglia cells were then isolated from the other glial cells using a cell sorter, as shown in Fig. 1A, and cultured in flat-bottom 96-well plates. CD11b+ microglial cells could efficiently induce activation and proliferation of resting A{beta}-reactive T cells in a dose-dependent fashion provided that the microglia were preincubated with IFN-{gamma} (Fig. 1B). In the absence of IFN-{gamma}, we did not observe T cell activation as measured by cytokine production or proliferation as measured by thymidine incorporation (data not shown). The microglial cells could stimulate T cell activation with both A{beta}1–40 and A{beta}1–42 (Fig. 1B). A{beta}1–42 has been shown to be more aggregative and cytotoxic than A{beta}1–40, and an increase in A{beta}1–42 has been associated more than that of A{beta}1–40 with AD progression. To test whether microglia-mediated T cell proliferation was dependent on CD80/CD86-CD28 or CD40-CD40L costimulatory pathways, we added CTLA-4-Ig or anti-CD40L to the culture at the time of stimulation. T cell proliferation was efficiently blocked in the presence of CTLA-4-Ig at all Ag concentrations tested (p < 0.001) but not in the presence of anti-CD40L (Fig. 1B). Of note, at the higher concentrations (50 µg/ml) of A{beta}1–40 T cell proliferation was slightly increased in the presence of anti-CD40L (Fig. 1B) although this was not statistically significant.



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  		FIGURE 1. IFN-{gamma}-treated microglia induce CD86-dependent proliferation of A{beta}-reactive T cells. Primary glial cultures were incubated with IFN-{gamma} at day 7 for 72 h. Cultures were then trypsinized and whole glial cells were removed, washed, and prepared for extracellular staining. Cells were stained with FITC-labeled anti-CD11b, and positive cells were sorted using FL-1 (FITC) and FL-2 (PE) detectors (A). Microglial cells were then cultured in 96-well plates (2 x 104/well) and 2 h later resting A{beta}-reactive T cells (5 x 104/well) were added to the wells in the presence or absence of human A{beta}1–40 or A{beta}1–42. A{beta}-reactive T cell proliferation was measured at 72 h after stimulation, as described in Materials and Methods. Microglia-mediated T cell proliferation was measured in the presence of CTLA4-Ig (10 µg/ml; B), anti-CD40L (10 µg/ml; B), anti-CD80 (10 µg/ml C), and anti-CD86 (10 µg/ml; C) at various A{beta} concentrations. Note that synthetic crude forms of A{beta} peptides were used in B and commercial synthetic forms (BioSource International) were used in C and in the other experiments described.

 
CD80 or CD86 costimulatory molecules are major costimulatory molecules for T cell activation in the CNS with a differential effect on pathogenicity (30, 31, 32). Thus, we further explored whether the blocking of T cell proliferation by CTLA4-Ig was related to CD80, CD86, or both by adding Abs to these costimulatory molecules to the culture at the time of stimulation. Only anti-CD86 Ab blocked T cell proliferation (p < 0.001) (Fig. 1C), although both CD80 and CD86 were up-regulated on the CD11b+ microglia following IFN-{gamma} stimulation (data not shown).

CD11b+ microglia induce the proliferation of A{beta}-reactive Th2 as compared with Th1 cells following A{beta} stimulation

To test whether microglia differ with regard to stimulation of Th1- and Th2-type T cells, we generated A{beta}-reactive Th1 and Th2 cell lines and cultured them with IFN-{gamma}-treated microglia. We first established the characteristics of the cell lines with irradiated splenocytes as APCs. When stimulated by an increasing number of irradiated splenocytes, the rates of proliferation of Th1 and Th2 cells were similar, with a plateau reached at 2.5 x 106 cells/ml (Fig. 2A, top panel). In addition, as expected, activation of Th1 cells resulted in increased secretion of IFN-{gamma} (Fig. 2A, middle panel), whereas activation of Th2 cells induced the expression of IL-10 (Fig. 2A, bottom panel).



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  		FIGURE 2. CD11b+ microglia suppress proliferation of Th1 cells following their stimulation. Primary glial cultures were treated with IFN-{gamma} at day 7 for 72 h and CD11b+ cells were sorted as described in Materials and Methods. Th1 and Th2 A{beta}-reactive T cells were prepared as described in Materials and Methods. T cells (5 x 104/well) were cocultured with increasing numbers of either syngenic irradiated splenocytes (A) or CD11b+ microglia (B) in the presence of 10 µg/ml A{beta}1–42. T cell proliferation and cytokine production were measured at 72 and 40 h after stimulation, respectively. Cytokines were measured by ELISA as described in Materials and Methods. Microglial cells were also cocultured with A{beta}-reactive T cells in culture slides (as described in Materials and Methods) in the presence or absence of A{beta} and analyzed immunocytochemically for IL-10 expression. IL-10 staining was observed only after T cell stimulation by microglia in the presence of A{beta} (C).

 
To test activation of Th1 vs Th2 cells by microglia, we sorted CD11b+ cells as described in Fig. 1 and cultured an increasing number of cells in flat-bottom 96-well plates until adherence could be observed. Resting Th1 and Th2 A{beta}-reactive T cells were then added to the culture along with 10 µg/ml A{beta}1–42. As shown in Fig. 2B (top panel), CD11b+ microglia supported Th2 cell proliferation equally as well as irradiated splenocytes, whereas there was markedly reduced proliferation of the Th1 cells cultured with CD11b+ microglia. However, as shown in Fig. 2B (middle panel), CD11b+ microglia, like splenocytes, supported Th1 cell secretion of IFN-{gamma}. When we examined IL-10, we found that CD11b+ microglia differed from splenocytes in that IL-10 secretion was observed in cultures of Th1 cells stimulated by CD11b+ microglia (Fig. 2B, bottom panel), whereas IL-10 was not detected in cultures of Th1 cells stimulated with irradiated splenocytes, (Fig. 2A, bottom panel). Furthermore, greater amounts of IL-10 were produced in cultures of T cells plus CD11b+ microglial cells than in cultures of T cells with irradiated splenocytes (Fig. 2, A and B). This raised the possibility that the increased levels of IL-10 seen in the T cell microglial cultures (Fig. 2B, bottom panel) might be related to the production of IL-10 by microglia. To test this possibility, we immunolabeled microglia with fluorescent anti-IL-10 Abs 72 h after treatment with IFN-{gamma} and coculture with A{beta}-reactive T cells in the presence of A{beta}. As shown in Fig. 2C, intracellular staining of IL-10 was detected in microglia after culture with either Th1 or Th2 cells. This was not observed in IFN-{gamma}-treated microglia that were not cultured with T cells (data not shown), suggesting that stimulation of T cells was required for IL-10 production by microglia.

To further investigate our observation that microglia did not support proliferation of Th1 cells (Fig. 2B, top panel), we investigated cytokine expression in microglia Th1/Th2 cultures at 24 h after stimulation. We found increased production of IL-4, IL-10, TNF-{alpha}, and GM-CSF in Th2 microglia cultures as compared with Th1 microglia cultures, A{beta} microglia cultures, or microglia cultured alone (Fig. 3). Stimulation of Th1 cells by microglia resulted in secretion of substantial amounts of IL-10, TNF-{alpha}, and higher levels of IFN-{gamma} (Fig. 3). Although the microglia were preactivated with IFN-{gamma}, none of the cytokines was expressed by microglia when cultured alone or in the presence of A{beta} (Fig. 3), suggesting that T cell stimulation was required for microglia to produce IL-10 and TNF-{alpha}.



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  		FIGURE 3. Cytokine profile of Th1 and Th2 A{beta}-reactive T cells 24 h after stimulation by microglia. CD11b+ microglia were isolated as described in Materials and Methods and 2 x 104 cells/well were cultured in flat-bottom 96-well plates. After their adherence, microglia were supplemented with medium only or A{beta} with or without A{beta}-reactive T cells (5 x 104/well). Supernatants were collected 24 h after stimulation and cytokines were measured by ELISA.

 
NO inhibitor restores microglia-mediated proliferation of A{beta}-reactive Th1 cells

Exposure of microglia and astrocyte cells to A{beta} has been found to increase levels of NO (25, 33). Increased mRNA levels of iNOS and argininosuccinate synthetase also were detected in brain tissue of patients with AD (34, 35), suggesting a role for NO in A{beta} pathogenicity. To elucidate the role that NO may play in the microglia T cell cultures, we first measured the levels of NO produced in the microglia culture alone in the presence and absence of A{beta} and 48 and 72 h following T cell stimulation by microglia. At 48 h, A{beta} alone did not induce increased levels of NO by microglia; however, a greater than 2-fold increase was observed in the Th1 microglia culture as compared with a 1.4-fold increase in the Th2 microglia culture (Fig. 4A). At 72 h after stimulation, NO levels also were increased by microglia in the presence of A{beta} alone and were further increased only in the Th1 microglia culture (Fig. 4A).



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  		FIGURE 4. Inhibition of Th1 cell proliferation is mediated via secretion of NO following Ag presentation to Th1 cells. CD11b+ microglia were sorted from IFN-{gamma}-treated glial cells and cultured in flat-bottom 96-well plates (2 x 104/well). Microglia were then incubated with and without 10 µg/ml A{beta}1–42 in the presence and absence of A{beta}-reactive T cells (5 x 104/well). A, Supernatants were collected at 40 and 72 h after stimulation and NO levels were measured according to the manufacturer’s instructions (R&D Systems). B, Th1 cell proliferation was measured 72 h after stimulation in the presence or absence of 1 mM of the iNOS inhibitor L-NIL. C, Th1 cell proliferation was measured 72 h after stimulation in the presence of increased concentrations of L-NIL. D, Th1 cell proliferation also was measured after stimulation by microglia in the presence of Abs to IL-10 (10 µg/ml), IFN-{gamma} (10 µg/ml), and Fas-L (25 µg/ml) according to the manufacturer’s instructions.

 
To test whether iNOS blockage could restore the proliferation of Th1 cells, we added the iNOS inhibitor L-NIL to the culture at the time of T cell stimulation. Th1 proliferation measured 72 h after stimulation was efficiently restored in the presence of 1 mM L-NIL (Fig. 4B). iNOS inhibition mediated the restoration of Th1 cell proliferation in a dose-dependent fashion (Fig. 4C). The inhibition of Th1 cell proliferation by microglia could involve cytokine expression such as IL-10 and IFN-{gamma} in addition to being mediated by Fas-Fas-L interaction. Th1 cell proliferation was therefore tested in the presence of neutralizing Abs to IL-10, IFN-{gamma}, and Fas-L which were added to the culture at the time of T cell stimulation. We found that none of the Abs restored the proliferation of A{beta}-reactive Th1 cells (Fig. 4D).

To determine whether A{beta} induced iNOS expression in microglial cells and to determine whether the expression of iNOS was increased when Th1 or Th2 cells were cultured with CD11b+ microglial cells, we conducted immunocytochemistry analysis of iNOS after microglia incubation with A{beta}1–42 plus Th1 or Th2 cells. As shown in Fig. 5, there was an increase in iNOS expression by microglia at 72 h after incubation with A{beta}1–42 (Fig. 5, top panels). When we then added Th1 or Th2 cells to the microglia A{beta} cultures, we observed an increase in iNOS expression after microglia incubation with A{beta}1–42 and Th1 cells that was significantly more than that observed with Th2 cells (Fig. 5, lower panels). These results are consistent with Fig. 4A.



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  		FIGURE 5. Regulation of A{beta}-induced iNOS expression by microglia after stimulation of Th1 and Th2 A{beta}-reactive T cells. CD11b+ microglia were sorted as described in Materials and Methods and cultured alone (2 x 105/ml) or in the presence of A{beta}-reactive Th1 and Th2 cells (5 x 105/ml) in the presence of 10 µg/ml A{beta}1–42. iNOS staining was performed 72 h after T cell stimulation as described in Materials and Methods and images were taken by confocal microscopy. Each panel contains two figures: the square figure in the left part of each panel demonstrates immunostaining of iNOS-expressing microglia and the figure in the right part of each panel demonstrates staining intensity of individual cells by confocal three-dimensional analysis. The x and y horizontal axes represent the area of individuals cells in micrometers and the vertical z-axis represents fluorescent intensity.

 
Microglia induce apoptosis of Th1 cells following costimulation

Microglia suppressed the proliferation of A{beta}-reactive Th1 cells following stimulation, whereas A{beta}-reactive Th2 cells proliferated efficiently. To determine whether inhibition of A{beta}-reactive Th1 cell proliferation was followed by apoptosis or cell arrest, we first took images of live Th1 and Th2 cultures 72 h after their stimulation by microglia. In the absence of A{beta}, microglia cells maintained an activated morphology and elongated processes and T cells remained resting (Fig. 6, a and b). In the presence of A{beta}, although activated by microglia, the Th1 culture appeared not to proliferate (Fig. 6c); Th1 cells were enlarged and showed signs of early apoptosis adjacent to vacuole-containing microglia (Fig. 6c, arrows). Th2 cultures showed activated T cells with filiopodia formation and clusters of proliferating cells adjacent to dendrite-forming microglia (Fig. 6d, arrows). Cell density was significantly higher in the Th2 microglia culture as compared with the Th1 microglia culture as a result of Th2 cell proliferation (Fig. 6, c and d).



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  		FIGURE 6. Th1 cells undergo apoptosis following stimulation by CD11b+ microglia. CD11b+ microglia were sorted as described in Materials and Methods and cocultured (2 x 105/ml) with A{beta}-reactive T cells (5 x 105/ml) in the presence or absence of 10 µg/ml A{beta}1–42. Images were taken 72 h after stimulation using an Olympus CK40 microscope equipped with Olympus digital camera DP-12 (Olympus, Melville, NY). Images were magnified x 100. Arrows point to T cells (T) and microglia (M).

 
We then measured levels of active caspase-3 on days 2 and 3 after stimulation as an indicator of T cell apoptosis. Th1 and Th2 cells were harvested from the plate and intracellular levels of active caspase-3 were measured by FACS analysis. Active caspase-3 could not be detected 40 h after stimulation (Fig. 7). On day 3 after stimulation, 30% of the CD4-labeled Th1 cells, as compared with 4% of the Th2 cells, were stained for caspase-3 (Fig. 7). The addition of NO inhibitor reduced the percentage of Th1 cells expressing active caspase-3 to 8%. The Th1 caspase-3-negative cells stained less intensively for CD4 in the absence of iNOS inhibitor than in its presence or than staining at 40 h after stimulation (Fig. 7), suggesting that they were nonactivated cells. It should be noted that a ratio of T cells to microglia of 5:1 was used, and thus presumably most of the activated Th1 cells already were undergoing apoptosis at this time point.



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  		FIGURE 7. Increased levels of active caspase-3 in A{beta}-reactive Th1 cells stimulated by microglia. CD11b+ microglia were sorted as described in Fig. 1 and cocultured with A{beta}-reactive T cells in the presence of 10 µg/ml A{beta}1–42. T cells were harvested at 40 and 72 h after stimulation and stained with FITC-labeled anti-CD4 followed by intracellular staining with PE-labeled anti-active caspase-3 as described in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We found that microglia were efficient A{beta} APCs providing that they were pretreated with IFN-{gamma}. A{beta}-reactive T cell activation was CD86 dependent but not CD80 dependent, although both were up-regulated in the presence of IFN-{gamma}. CD11b+ microglia, although they induced cytokine secretion, failed to support the proliferation of A{beta}-reactive Th1 cells. Th1 cells underwent NO-mediated apoptosis after their stimulation by activated microglia.

Many studies have noted profound T cell death in the CNS during inflammatory cascades and have attributed it primarily to Fas-Fas-L interactions, as glial cells express relatively high levels of Fas/Fas-L (2, 9, 10). Fas and Fas-L have been found to be up-regulated in IFN-{gamma}-stimulated microglia cell lines, implicating a role for IFN-{gamma}-induced cell death via Fas-Fas-L interactions (36). In the present study, Fas-blocking Abs did not restore Th1 cell proliferation, although microglia were pretreated with IFN-{gamma}, which also was produced at high levels during T cell stimulation.

Coculture of IFN-{gamma}-treated microglia with Th1 cells resulted in the secretion of high levels of IFN-{gamma} and IL-10 followed by T cell apoptosis. Proliferation of A{beta}-reactive Th1 cells could be restored in the presence of iNOS inhibitor, suggesting a role for NO secretion. Stimulation of T cells with irradiated splenocytes did not decrease proliferation of A{beta}-reactive Th1 cells although IFN-{gamma} levels induced were similar to those induced in the Th1 microglia culture. An increasing body of evidence has shown that macrophages can suppress T cell proliferation via NO production, for which IFN-{gamma} was essential but not always sufficient (37, 38, 39, 40). As shown in the present study, pretreatment of microglia with IFN-{gamma} was not sufficient to suppress T cell proliferation and neutralizing Abs to IFN-{gamma} did not restore T cell proliferation. However, the increased levels of NO induced by A{beta} alone were further increased by IFN-{gamma} and in the absence of Th2 cytokines promoted T cell apoptosis.

A{beta}, specifically A{beta}1–42, is toxic to neurons and is hence strongly associated with loss of brain function through the course of AD (19). This deposition induces migration and activation of astrocytes and microglia that further mediate neuronal cytotoxic pathways (20). A{beta} plaques therefore become chronic lesions in the brain that damage the neighboring neurons. Extensive studies in recent years have focused on A{beta}-mediated microglial activation with regard to secretion of proinflammatory cytokines such as TNF-{alpha} and IL-1{beta}, activation of complement factors, and release of free radicals (20, 21, 35, 41, 42). However, the possibility of A{beta} uptake, processing, and presentation in the context of MHC has not been characterized in AD. In the present study, both A{beta}1–40 and the more aggregative form, A{beta}1–42, could be processed by stimulated microglia to support activation and proliferation of Th2-type A{beta}-reactive T cells. Therefore, with regard to A{beta}, this pathway could have an important physiologic role, perhaps by contributing to enhanced clearance of A{beta} and also through bystander down-regulation of the proinflammatory milieu. However, as NO production is constitutively induced at sites of A{beta} plaques (25, 33, 34, 35), T cells may undergo apoptosis before their activation by microglia. In this environment, microglia become activated and contribute to a chronic innate immune response in the CNS with very limited capacity as APCs.

NO cytotoxicity has recently been characterized as playing an important role in models of experimental allergic encephalomyelitis (EAE). EAE severity was increased in iNOS knockout animals (43, 44). Oral administration of N-methyl-L-arginine acetate (an iNOS inhibitor) to Lewis rats after their recovery from EAE resulted in a chronic relapsing disease (45). Therefore, NO appears to play a key role in immune regulation. IFN-{gamma} plays a central role in the initiation of Th1 responses in the CNS, including the induction of iNOS. However, because of the synthesis of NO, T cells that are stimulated by microglia to secrete cytokines fail to proliferate and undergo apoptosis. As shown in the present study, Th1 stimulation by microglia also induced high levels of IL-10, which can down-regulate NO through down-regulation of iNOS and up-regulation of arginase I or II (46). Levels of NO can further decrease in the presence of Th2 and regulatory cytokines such as IL-4, IL-13, and TGF-{beta} that also down-regulate iNOS and up-regulate arginase (46). In the present study, NO was up-regulated by A{beta} in the Th2 culture; however, the predominant presence of Th2 cytokines as early as 24 h after stimulation counterbalanced the levels of NO and promoted activation and proliferation of Th2 cells. As recently demonstrated, a minor reduction in NO levels appeared to be essential to allow T cell proliferation (38). A delicate balance of NO levels may therefore play an important regulatory role in switching from Th1 to Th2 immune responses in the CNS.

IFN-{gamma}, in addition to its role in induction of iNOS, is one of the most potent stimulators of microglia as APCs (47). In the present study, pre-exposure of microglia to IFN-{gamma} was required for A{beta} presentation in the context of MHC class II, as without it there was no activation of A{beta}-reactive T cells. IFN-{gamma}, however, is a predominant Th1 cytokine that has not been shown to be expressed in the CNS of adult animals in situ. Thus, upon infiltration of IFN-{gamma}-producing cells (primarily T and NK cells), microglia undergo full maturation as APCs, which presumably occurs rarely in parenchymal regions of the CNS (48). GM-CSF also has been shown to be a potent differentiating factor of microglia as APCs (15, 16, 17, 49), and as demonstrated in the present study, its expression in the microglia Th2 culture probably increased the potency of microglia as APCs. Of interest is the finding that GM-CSF levels in the cerebrospinal fluid of patients with AD or vascular dementia are markedly increased (50). Thus, in AD microglial cells have the potential to differentiate into APCs in the CNS and interact with infiltrating T cells and this may relate to the increased occurrence of T cells observed in patients with AD as compared with age-matched controls or patients with other neurodegenerative diseases (51).

Immunization of amyloid precursor protein transgenic mice with A{beta}-CFA has been found to be very effective in clearance of A{beta} from the CNS (27). We also found that nasal administration of A{beta} decreased A{beta} plaque burden in the CNS and was accompanied by infiltration of TGF-{beta}-, IL-10-, and IL-4–secreting T cells into the CNS (28). TGF-{beta} and IL-10 have been shown to protect neurons from a variety of insults such as A{beta}-induced apoptosis, down-regulation of NO, and stroke (52, 53). Our results suggest that microglia can be modulated to play a beneficial role in AD. Their modulation depends primarily on the cytokine milieu generated at sites of A{beta} plaques and can be significantly influenced by the type of T cells induced (54). Thus, microglia-mediated secretion of NO may play a beneficial role in EAE by inducing Th1 cell apoptosis following stimulation of Th1 cells. However, its constitutive expression at sites of A{beta} plaques may suppress T cell activation and contribute to chronic neurotoxicity.


   Acknowledgments
 
We thank Dr. Vijay Kuchroo for his comments on this manuscript and Rob McGilp for sorting the cells.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants NS 38037-01A1 and RO1 AI 435801. Back

2 Address correspondence and reprint requests to Dr. Alon Monsonego, Center for Neurologic Diseases, Harvard Medical School, Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, HIM 730, Boston, MA 02115-5817. E-mail address: amonsonego{at}rics.bwh.harvard.edu Back

3 Abbreviations used in this paper: FasL, Fas ligand; A{beta}, amyloid {beta}-peptide; AD, Alzheimer’s disease; EAE, experimental allergic encephalomyelitis; iNOS, inducible NO synthase; L-NIL, L-N6-(1-iminoethyl)-lysine · 2 HCl. Back

Received for publication November 27, 2002. Accepted for publication June 19, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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