- Email: [email protected]
Microglia phagocytose alloreactive CTL-damaged 9L gliosarcoma cells
Journal of Neuroimmunology 153 (2004) 76 – 82 www.elsevier.com/locate/jneuroim
Microglia phagocytose alloreactive CTL-damaged 9L gliosarcoma cells Nisha V. Kulprathipanja a, Carol A. Kruse a,b,* a
Department of Immunology, University of Colorado Health Sciences Center, 4200 E. 9th Avenue, B216, Denver, CO 80262, USA b Department of Pathology, University of Colorado Health Sciences Center, Room 2653 Campus Box B216, 4200 E. Ninth Avenue, Denver, CO 80262, USA Received 15 March 2004; received in revised form 21 April 2004; accepted 21 April 2004
Abstract Intracranial adoptive transfers of alloreactive cytotoxic T lymphocytes (aCTL) for brain tumor treatment were safe and showed promise in preclinical and early clinical trials. To better understand the endogenous immune responses that may ensue following cellular therapy with aCTL, we examined the ability of microglia to phagocytose aCTL-damaged and undamaged rat 9L gliosarcoma cells in vitro and in vivo. In vitro, 5.5 F 0.9% of microglial cells isolated from adult tumor-bearing rat brains phagocytosed aCTL-damaged 9L cells, whereas microglia did not bind to or ingest undamaged 9L cells. Addition of supernates from either 9L cell cultures or from aCTL + 9L co-incubate cell cultures to microglia did not significantly alter their ability to bind to or phagocytose damaged glioma cells even though the latter contained T helper 1 and 2 cytokines. At 3 days following intracranial 9L cell infusion, 17.5 F 0.1% of the microglia phagocytosed CFSE-labeled aCTL-damaged 9L tumor cells within the adult rat brain, confirming the in vitro data. The results suggest that microglia within the tumor microenvironment of the adult rat glioma model selectively remove damaged, but not undamaged, glioma cells. D 2004 Elsevier B.V. All rights reserved. Keywords: Microglia; Phagocytosis; Glioma; Antigen presentation; Apoptosis; CTL
1. Introduction The primary goal of therapies for malignant gliomas is to induce tumor cell death. However, the role(s) that microglia may play in the fate of the damaged glioma cells is an issue that has not been extensively investigated. Microglia have been observed in high numbers in human brain tumors (Graeber et al., 2002; Lorusso and Rossi, 1997) and in experimental rat glioma models (Badie and Schartner, 2000; Morioka et al., 1992). Because microglial cells derived from neonatal rodent and human brains phagocytose neuronal and myelin debris (Magnus et al., 2002; Smith, 2001; Streit et al., 1999; Witting et al., 2000), it is likely that microglial cells are involved in the clearance of damaged glioma cells within the brain. In vitro studies by Chang et al. (2000) support this idea by demonstrating the ability of human microglia to phagocytose apoptotic glioma cells. * Corresponding author. Present address: La Jolla Institute for Molecular Medicine, 4570 Executive Drive, Ste 100, San Diego, CA 92121, USA. Tel.: +1-858-587-8788x142; fax: +1-858-587-6742. E-mail address: [email protected] (C.A. Kruse). 0165-5728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2004.04.011
Our laboratory has been studying cellular immune therapies that have the potential to selectively destroy malignant brain tumor cells while leaving normal brain cells unharmed. We have shown that one specific type of effector cell population, alloreactive cytotoxic T lymphocytes (aCTL) sensitized to the major histocompatibility complex (MHC) antigens of the tumor host, was tolerable and showed promise when administered intracranially in preclinical and clinical studies (Fleshner et al., 1992; Kruse et al., 1997, 1990, 1994b; Kruse and Rubinstein, 2001; Read et al., 2003). Therefore, we are particularly interested in the interaction of other immune cells, such as microglia, with glioma cells that have been damaged by aCTL treatment. Information on whether microglial cells are capable of removing damaged gliomas undergoing apoptosis or necrosis is important for designing treatments that minimize damage to normal brain tissue and inflammatory processes in the brain. In this study, we investigated the ability of microglia/macrophages isolated from tumor-bearing adult rat brains to phagocytose undamaged, ultraviolet radiation (UV)-damaged, and aCTL-damaged 9L glioma cells in vitro and in vivo.
N.V. Kulprathipanja, C.A. Kruse / Journal of Neuroimmunology 153 (2004) 76–82
2. Materials and methods 2.1. Cell culture, antibodies, and reagents The rat 9L gliosarcoma cell line was maintained in RPMI-1640 and Dulbecco’s Modified Eagle’s Medium (DMEM) (2:1 vol/vol) supplemented with 10% fetal bovine serum (FBS), 200 U/ml penicillin, and 200 Ag/ml streptomycin (Abs). Microglia were maintained in DMEM with 10% FBS and Abs. aCTL were cultured in Iscove’s Modified Dulbecco’s Medium supplemented with 10% FBS, 2 mM L-glutamine, 50 AM 2-mercaptoethanol, 0.5 mM N-monomethyl-L-arginine-HOAc (L-NMMA, CBC Cyclopss Biochemical, Salt Lake City, UT), 50 Ag/ml gentamicin, and Abs. Cells were kept in 5% CO2 at 37 jC in a humidified incubator. Mouse anti-rat CD11bphycoerythrin (PE), mouse anti-rat CD11b-allophycocyanin (APC), mouse IgG2a-APC isotype control, and annexin-V-APC were purchased from Caltag Laboratories, Burlingame, CA. The mouse IgG2a-PE isotype control was purchased from Beckman Coulter, Fullerton, CA. Propidium iodide (PI) was from R&D Systems, Minneapolis, MN. Carboxy-fluorescein diacetate, succinimidyl ester (CFSE) was from Molecular Probes, Eugene, OR, and 7amino actinomycin D (7-AAD) was from ICN Biomedicals, Irvine, CA.
77
er lymphocytes were then mixed at a 1:1 ratio in a one-way mixed lymphocyte reaction, and the aCTL were used after 5 –7 days in culture. 2.5. Isolation of aCTL-damaged, UV-damaged, and undamaged 9L cells aCTL and 9L cells (pre-labeled with 0.5 AM CFSE according to manufacturer’s protocol) were co-incubated at an effector to target ratio of 10:1 for 18 –24 h at 37 jC in a 5% CO2 humidified chamber. The supernate was then collected and clarified (12,500 gmin) for use in the phagocytosis assays. The cells were harvested and labeled with 200 Ag/ml 7-AAD. After washing with PBS, a portion of the cells was stained with the phosphatidylserine (PS) early apoptosis marker, annexin-V-APC, and with PI. The aCTL-damaged glioma cells (CFSE and 7-AAD positive) were then sorted from the remaining cells using a flow cytometer (MoFlo Cell Sorter, DakoCytomation, Fort Collins, CO). Damaged 9L cells that had detached from the culture flask 30 h after a 10-min exposure to 40 AW/cm2 UV and untreated 9L cells were added as the controls in the phagocytosis assay. The UV-damaged and undamaged 9L cells were also labeled with CFSE prior to use in the assays, and their damage profiles were assessed using 7-AAD and annexin-V-APC/PI flow cytometric assays.
2.2. Intracranial infusion of tumor 2.6. In vitro phagocytosis assay 9L cells were intracranially implanted into anesthetized adult (6 –8 weeks) F344 rats as described before (Kruse et al., 1990). Two infusions of 9L cells (5 105 cells/10 Al) in phosphate buffered saline (PBS) were infused at a depth of 4 mm below the dura using a 50-Al Hamilton syringe through small burr holes at 3 mm to the right and to the left of the midline, 2 mm anterior to the bregma. Microglia were recovered from brain tissue 7– 10 days post-tumor infusion. 2.3. Isolation of microglia Microglia were isolated as described by Ford et al. (1995) with minor modifications. Briefly, brains from normal adult (6– 8 weeks) rats or rats bearing intracranial 9L tumors were minced with scissors and mechanically dissociated with a glass homogenizer. The microglia were collected using two discontinuous Percoll (Amersham Biosciences, Piscataway, NJ) gradients and were washed twice before use.
Microglia were added to 24-well plates at 2 105 cells/ well and were allowed to adhere to the plates for 2 h. CFSE-labeled aCTL-damaged, UV-damaged, or undamaged 9L glioma cells were added to the microglial monolayers for 3 h. In select wells, microglial cells were placed in 24-h supernate from either 9L + aCTL co-cultures or from undamaged 9L cultures during the phagocytosis assay. Cytochalasin D (5 Ag/ml final concentration, Sigma, St. Louis, MO), which prevents phagocytic function by inhibiting actin cytoskeleton polymerization, was added to one set of control wells containing microglia 30 min prior to the addition of 9L cells (Jersmann et al., 2003). The cells were harvested, stained with CD11b-APC, and fixed in 1% formaldehyde in PBS. Cells were analyzed using a flow cytometer (BD FACSCalibur, Becton Dickinson, San Jose, CA). Phagocytosis was evaluated by subtracting the background binding observed in the control wells from the percentage of CD11b+ microglia also positive for CFSE in the experimental wells.
2.4. Generation of aCTL 2.7. In vivo phagocytosis assay aCTL were prepared and isolated as described before (Paul et al., 2003). F344 rats (6– 8 weeks) were the source of stimulator lymphocytes and DA or Sprague – Dawley rats (6– 8 weeks) were the source of responder lymphocytes. Stimulator (60Co-gamma irradiated at 2500 rad) and respond-
aCTL-damaged, UV-damaged, and undamaged 9L glioma cells labeled with 10 AM CFSE were intracranially implanted into adult rat brains as described above. Control rats were infused with aCTL-damaged, UV-damaged, or
78
N.V. Kulprathipanja, C.A. Kruse / Journal of Neuroimmunology 153 (2004) 76–82
Fig. 1. Flow cytometric analysis (left panel) of microglia from normal adult rat brains stained with CD11b-PE (open) and an isotype antibody (shaded). Microglia from tumor-bearing adult rat brains 2 h after plating as observed by light microscopy (right panel, bar = 10 Am).
undamaged 9L cells that were not CFSE labeled. Microglial cells were isolated from the rat brains at 3 or 5 days after glioma infusion and were stained with CD11b prior to analysis by flow cytometry. The gates for analyzing phagocytic microglial cells (CFSE positive) were set using the population of microglial cells from the control rats (CFSE negative).
3. Results 3.1. Microglial cell yields are higher from glioma-bearing brains than from normal adult rat brains Microglia freshly isolated from normal (Fig. 1, left panel) and 9L tumor-bearing adult rat brains expressed
CD11b on their cell surfaces, and the cells adhering to the plating surface primarily displayed an amoeboid morphology (Fig. 1, right panel). Approximately two-thirds of the CD11b+ cells were of the CD45low microglial phenotype (data not shown), implicating the remaining CD11b+ cells as CNS macrophages (Ford et al., 1995). For our assays, all CD11b+ cells were used. The microglial cell yields from intracranial tumor-bearing rat brains were approximately 10-fold higher, at 14.6 104 to 37.8 104 cells/rat (average 29.5 104), than from normal brains which ranged from 1.4 104 to 5.4 104 cells/brain (average 2.7 104). Cell viabilities of freshly isolated microglia, as assessed by trypan blue dye exclusion, were 70% to 95% (n = 15). Similar microglial cell yields (data not shown) were obtained from rats bearing CNS-1 gliomas (Kruse et al., 1994c). 3.2. 9L cells are injured upon exposure to aCTL or UV radiation 9L cell injury was confirmed by flow cytometric analysis of 7-AAD and annexin-V/PI staining. The majority (87 – 94%) of untreated 9L cells were viable (Fig. 2A and D). However, 96% of UV-exposed and 66% of aCTLexposed 9L cells incorporated 7-AAD into their DNA, reflecting a high percentage of injured cells in both populations (Fig. 2B and C). Moreover, 48% of both the UV-exposed and aCTL-exposed cells expressed the early apoptotic marker, annexin-V (Fig. 2E and F). 9L cells damaged by UV and aCTL exposure (Fig. 3C and D) also
Fig. 2. 9L glioma cell injury assessed by 7-AAD (top panels) and annexin-V-APC/PI (bottom panels) flow cytometric assays. Flow diagrams are shown for untreated (A, D), UV-exposed (B, E), and aCTL-exposed (C, F) 9L cells.
N.V. Kulprathipanja, C.A. Kruse / Journal of Neuroimmunology 153 (2004) 76–82
79
Fig. 3. Laser scatter profile of (A) microglial cells, (B) microglia and undamaged 9L cells, (C) microglia and UV-damaged 9L cells, and (D) microglia and aCTL-damaged 9L cells from an in vitro phagocytosis assay. The microglial cells, which are encircled by the oval gate in the dot-plot, were confirmed to express CD11b.
3.3. Microglial cells bind to and phagocytose UV-damaged and aCTL-damaged 9L, but not undamaged 9L cells in vitro
nates from 9L cell or aCTL + 9L cell cultures did not significantly alter the ability of the microglia to bind to or phagocytose glioma cells (data not shown), even though multiple cytokines (IFN-gamma, TNF-alpha, GM-CSF, IL6, and IL-10) were detected in the aCTL + 9L cell culture supernate, but not the 9L culture supernates (data not shown).
Microglial cells in the phagocytosis assays were easily identified by flow cytometry in forward scatter/side scatter dot-plots (Fig. 3, oval gates), and 60– 80% of the cells within the oval gates expressed CD11b (data not shown). The microglial cells displaying CD11b were then analyzed for CFSE positivity to assess the ability of microglia to bind/phagocytose CFSE-labeled 9L cells. The background binding was observed by blocking phagocytosis with cytochalasin D. 6.8 F 1.7% and 4.3 F 0.9% of the microglia bound to UV-damaged and aCTL-damaged 9L cells, respectively, but did not bind to undamaged 9L cells after a 3-h co-incubation (Fig. 4, black bars). Phagocytosis percentages were then calculated by subtracting the binding control percentages from the percentages of cells not exposed to cytochalasin D (Fig. 4, white bars). The microglia phagocytosed UV-damaged (8.1 F 0.4%) and aCTL-damaged (5.5 F 0.9%) 9L cells, but did not phagocytose undamaged 9L cells. Moreover, addition of super-
Fig. 4. The microglial cells positive for CD11b (oval gates, Fig. 3) were analyzed for CFSE positivity to indicate binding (cytochalasin D+) and binding plus phagocytic uptake (cytochalasin D-) of CFSE-labeled 9L cells in vitro. Microglia bind to (black bars) and phagocytose (white bars minus black bars) UV-damaged and aCTL-damaged, but not undamaged 9L glioma cells. Binding and phagocytosis from three independent experiments given as a percentage of CFSE-positive cells F S.E.M.
displayed decreased forward scatter profiles as compared to undamaged 9L cells (Fig. 3B), confirming the smaller cell sizes resulting from cell injury processes (Darzynkiewicz et al., 1997).
80
N.V. Kulprathipanja, C.A. Kruse / Journal of Neuroimmunology 153 (2004) 76–82
Fig. 5. In vivo microglial phagocytosis of undamaged, UV-damaged, and aCTL-damaged 9L glioma cells at days 3 and 5 after intracranial implantation. Phagocytosis from two independent experiments (2 rats/ group) given as a percentage of CFSE-positive cells F S.E.M.
3.4. Microglial cells phagocytose UV-damaged 9L and aCTL-damaged 9L, but not undamaged 9L cells in vivo Microglial cells were isolated and analyzed 3 and 5 days after intracranial implantation of either CFSE-labeled undamaged, UV-damaged, or aCTL-damaged 9L cells into adult rat brains (Fig. 5). Undamaged 9L cells were not phagocytosed by microglia in vivo at either 3 or 5 days. However, at 3-days post-implantation, 9.7 F 4.4% and 17.5 F 0.1% of the microglia phagocytosed CFSE-labeled UV-damaged and aCTL-damaged 9L cells, respectively. Few microglial cells were CFSE positive at 5 days, suggesting that the microglia degraded the ingested 9L cells by that time point.
4. Discussion Although cultured microglia from neonatal rat/mice brains are widely used in in vitro experiments, they are more activated than the resting, ramified microglia residing in adult rat brains (Becher and Antel, 1996; Carson et al., 1998, 1999). Therefore, microglial cells used in this study were isolated from adult tumor-bearing rat brains to more accurately reflect the activation state and phagocytic function of microglia present clinically in glioma patients. In addition, microglia were extracted from rat brains with an established tumor to better resemble the microglia found within glioma patients since the cells were likely exposed to a milieu of cytokines and chemokines within the tumor microenvironment, including several immunosuppressive factors (Fakhrai et al., 1996; Gillespie, 1996; Hao et al., 2002; Kiefer et al., 1994; Parney et al., 2000; Kielian et al., 2002; Read et al., 2003; Virasch and Kruse, 2001). As we and others (Badie and Schartner, 2000; Graeber et al., 2002; Lorusso and Rossi, 1997; Morioka et al., 1992) demonstrated, microglia are ubiquitous in glioma-bearing tissue. They are in close proximity to both healthy tumor cells and necrotic tumor cells, the latter being a feature of glioblastoma multiforme (Raza et al., 2002). Moreover, the
number of damaged glioma cells in juxtaposition with microglia increases after the administration of cytoreductive therapies that induce cell damage through apoptotic and necrotic pathways (Finkel, 1999; Gomez et al., 2004; Henkart et al., 1997; Jerome et al., 2003; Read et al., 2003). In particular, aCTL migrate from the instillation site through brain parenchyma (Kruse et al., 1994a), which increases the likelihood of tumor/effector cell contact, subsequent glioma cell damage, and microglial recognition of the damaged glioma cells. We have extensively studied intracranial adoptive transfer of aCTL into the brain preclinically and clinically (Fleshner et al., 1992; Kruse et al., 1997, 1990, 1994b; Kruse and Rubinstein, 2001). The responses noted may be a combined result of (1) direct interactions of the ex vivoactivated CTL effectors with the tumor cells, (2) cytokines secreted by the ex vivo-introduced effector CTL, (3) cytokines/chemokines produced by the glioma cells, and (4) interactions of endogenous immune cell components, including infiltrating microglial and T cells, with aCTL and glioma cells. Thus, analysis of microglial cell roles in aCTL cellular therapy is essential. The activation state of microglia after their interactions with damaged glioma cells, endogenous lymphocytes, adoptive therapeutic cells, and exposure to cytokines may have contradictory implications for the ability of the immune system to eliminate brain tumors (Badie and Schartner, 2001). Microglial immune functions are stimulated by pro-inflammatory T-helper 1 cytokines (Grau et al., 1997; Seguin et al., 2003), but are suppressed by anti-inflammatory T-helper 2 cytokines (Sawada et al., 1999). Phagocytosis of apoptotic cells has been shown to result in antiinflammatory responses (Hirt and Leist, 2003; Magnus et al., 2001; Witting et al., 2000), whereas exposure to necrotic debris results in pro-inflammatory responses (Fadok et al., 2001). Therefore, microglial phagocytosis of apoptotic gliomas may contribute to the existing array of immunoinhibitory factors within the tumor, which are also crucial for maintaining control over inflammation in the confined space of the brain. On the other hand, microglial uptake of damaged glioma cells may stimulate their secretion of proinflammatory signals, thereby maintaining microglia in an activated state. The activation state of microglia is crucial to their ability to present antigen to lymphocytes. We (unpublished data) and others (Badie and Schartner, 2000) demonstrated that microglia from tumor-bearing brains express major histocompatibility complex (MHC) class II and B7 costimulatory molecules that are necessary for antigen presentation. However, studies indicate the poor ability of neonatal microglia to present tumor antigen to T cells (Flugel et al., 1999; Taniguchi et al., 2000). The microenvironment surrounding aCTL-treated gliomas would likely contain high levels of pro-inflammatory cytokines and chemokines (IFN-gamma, TNF-alpha, and GM-CSF) since they were observed in supernates from rat and human aCTL/glioma cell co-incu-
N.V. Kulprathipanja, C.A. Kruse / Journal of Neuroimmunology 153 (2004) 76–82
bates (Read et al., 2003 and unpublished data). These cytokines may help maintain the activation state of microglial cells and increase their ability to present glioma antigens to T cells. In summary, we have confirmed that brains bearing tumors have higher infiltrates of microglia, making an assessment of their role there important. We confirm that aCTL treatment injured glioma cells and that the injury was sufficient for a small percentage of the microglial cells to recognize, bind to, and phagocytose the glioma cells both in vitro and in vivo. Microglial cell exposure to and uptake of injured glioma cells is likely an essential precursor to tumor antigen presentation. Therefore, microglia may play an important role in effecting the overall response observed with aCTL adoptive immunotherapy. Since activated microglia display some MHC Class I (Grau et al., 1997), they theoretically may be subject to aCTL damage. If the role of microglia as antigen-presenting cells is important in the overall therapy, their injury is not particularly worrisome, since they could be adoptively transferred into the brain subsequent to aCTL therapy. Further exploration in this model to assess the ability of adult microglial cells to present glioma cell antigen upon treatment with aCTL is warranted.
Acknowledgements This work was supported in part by National Institutes of Health (NIH) Grants RO1-NS56798 to CAK, and F31 NS 44074 to NVK and the R. Herbert and Alma S. Manweiler Memorial Research Fund.
References Badie, B., Schartner, J.M., 2000. Flow cytometric characterization of tumorassociated macrophages in experimental gliomas. Neurosurgery 46, 957 – 961. Badie, B., Schartner, J., 2001. Role of microglia in glioma biology. Microsc. Res. Tech. 54, 106 – 113. Becher, B., Antel, J.P., 1996. Comparison of phenotypic and functional properties of immediately ex vivo and cultured human adult microglia. Glia 18, 1 – 10. Carson, M.J., Reilly, C.R., Sutcliffe, J.G., Lo, D., 1998. Mature microglia resemble immature antigen-presenting cells. Glia 22, 72 – 85. Carson, M.J., Sutcliffe, J.G., Campbell, I.L., 1999. Microglia stimulate naive T-cell differentiation without stimulating T-cell proliferation. J. Neurosci. Res. 55, 127 – 134. Chang, G.H.F., Barbaro, N.M., Pieper, R.O., 2000. Phosphatidylserinedependent phagocytosis of apoptotic glioma cells by normal human microglia, astrocytes, and glioma cells. Neuro-Oncology 2, 174 – 183. Darzynkiewicz, Z., Juan, G., Li, X., Gorczyca, W., Murakami, T., Traganos, F., 1997. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 27, 1 – 20. Fadok, V.A., Bratton, D.L., Guthrie, L., Henson, P.M., 2001. Differential effects of apoptotic versus lysed cells on macrophage production of cytokines: role of proteases. J. Immunol. 166, 6847 – 6854. Fakhrai, H., Dorigo, O., Shawler, D.L., Lin, H., Mercola, D., Black, K.L., Royston, I., Sobol, R.E., 1996. Eradication of established intracranial
81
rat gliomas by transforming growth factor beta antisense gene therapy. Proc. Natl. Acad. Sci. U. S. A. 93, 2909 – 2914. Finkel, E., 1999. Biomedicine—does cancer therapy trigger cell suicide? Science 286, 2256 – 2258. Fleshner, M., Watkins, L.R., Redd, J.M., Kruse, C.A., Bellgrau, D., 1992. A 9L gliosarcoma transplantation model for studying adoptive immunotherapy into the brains of conscious rats. Cell Transplant 1, 307 – 312. Flugel, A., Labeur, M.S., Grasbon-Frodl, E.M., Kreutzberg, G.W., Graeber, M.B., 1999. Microglia only weakly present glioma antigen to cytotoxic T cells. Int. J. Dev. Neurosci. 17, 547 – 556. Ford, A.L., Goodsall, A.L., Hickey, W.F., Sedgwick, J.D., 1995. Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared. J. Immunol. 154, 4309 – 4321. Gillespie, G.Y., 1996. Cytokines as modulators of malignant glioma progression. In: Ransohoff, R.M., Benveniste, E.N. (Eds.), Cytokines and the CNS. CRC Press, Boca Raton, pp. 269 – 286. Gomez, G.G., Read, S.B., Gerschenson, L.E., Santoli, D., Zweifach, A., Kruse, C.A., 2004. Interactions of the allogeneic effector leukemic T cell line, TALL-104, with human malignant brain tumors. Neuro-Oncology 6, 83 – 95. Available at: http://neuro-oncology.mc.duke.edu. Graeber, M.B., Scheithauer, B.W., Kreutzberg, G.W., 2002. Microglia in brain tumors. Glia 40, 252 – 259. Grau, V., Herbst, B., VanderMeide, P.H., Steiniger, B., 1997. Activation of microglial and endothelial cells in the rat brain after treatment with interferon-gamma in vivo. Glia 19, 181 – 189. Hao, C.H., Parney, I.F., Roa, W.H., Turner, J., Petruk, K.C., Ramsay, D.A., 2002. Cytokine and cytokine receptor mRNA expression in human glioblastomas: evidence of Th1, Th2 and Th3 cytokine dysregulation. Acta Neuropathol. 103, 171 – 178. Henkart, P.A., Williams, M.S., Zacharchuk, C.M., Sarin, A., 1997. Do CTL kill target cells by inducing apoptosis? Semin. Immunol. 9, 135 – 144. Hirt, U.A., Leist, M., 2003. Rapid, noninflammatory and PS-dependent phagocytic clearance of necrotic cells. Cell Death Differ. 10, 1156 – 1164. Jerome, K.R., Sloan, D.D., Aubert, M., 2003. Measurement of CTL-induced cytotoxicity: the caspase 3 assay. Apoptosis 8, 563 – 571. Jersmann, H.P.A., Ross, K.A., Vivers, S., Brown, S.B., Haslett, C., Dransfield, I., 2003. Phagocytosis of apoptotic cells by human macrophages: analysis by multiparameter flow cytometry. Cytometry Part A 51A, 7 – 15. Kiefer, R., Supler, M.L., Toyka, K.V., Streit, W.J., 1994. In-situ detection of transforming growth-factor-beta messenger-RNA in experimental rat glioma and reactive glial-cells. Neurosci. Lett. 166, 161 – 164. Kielian, T., van Rooijen, N., Hickey, W.F., 2002. MCP-1 expression in CNS-1 astrocytoma cells: implications for macrophage infiltration into tumors in vivo. J. Neuro-Oncol. 56, 1 – 12. Kruse, C.A., Rubinstein, D., 2001. Cytotoxic T-lymphocytes reactive to patient major histocompatibility complex proteins for therapy of brain tumors. In: Liau, L.M., Becker, D.P., Cloughesy, T.F., Bigner, D.D. (Eds.), Brain Tumor Immunotherapy. Humana Press, Totowa, NJ, pp. 149 – 170. Kruse, C.A., Lillehei, K.O., Mitchell, D.H., Kleinschmidt-DeMasters, B., Bellgrau, D., 1990. Analysis of Interleukin-2 and various effector cellpopulations in adoptive immunotherapy of 9L rat gliosarcoma-allogeneic cytotoxic T-lymphocytes prevent tumor take. Proc. Natl. Acad. Sci. U. S. A. 87, 9577 – 9581. Kruse, C.A., Kong, Q.Z., Schiltz, P.M., Kleinschmidt-DeMasters, B.K., 1994a. Migration of activated lymphocytes when adoptively transferred into cannulated rat-brain. J. Neuroimmunol. 55, 11 – 21. Kruse, C.A., Schiltz, P.M., Bellgrau, D., Kong, Q.Z., Kleinschmidt-DeMasters, B.K., 1994b. Intracranial administrations of single or multiple source allogeneic cytotoxic T-lymphocytes. Chronic therapy for primary brain-tumors. J. Neuro-Oncol. 19, 161 – 168. Kruse, C.A., Molleston, M.C., Parks, E.P., Schiltz, P.M., KleinschmidtDeMasters, B.K., Hickey, W.F., 1994c. A rat glioma model, CNS-1
82
N.V. Kulprathipanja, C.A. Kruse / Journal of Neuroimmunology 153 (2004) 76–82
with invasive characteristics similar to those of human gliomas—a comparison to 9L gliosarcoma. J. Neuro-Oncol. 22, 191 – 200. Kruse, C.A., Cepeda, L., Owens, B., Johnson, S.D., Stears, J., Lillehei, K.O., 1997. Treatment of recurrent glioma with intracavitary alloreactive cytotoxic T lymphocytes and interleukin-2. Cancer Immunol. Immunother. 45, 77 – 87. Lorusso, L., Rossi, M.L., 1997. The phagocyte in human gliomas. Ann. N. Y. Acad. Sci. 832, 405 – 425. Magnus, T., Chan, A., Grauer, O., Toyka, K.V., Gold, R., 2001. Microglial phagocytosis of apoptotic inflammatory T cells leads to down-regulation of microglial immune activation. J. Immunol. 167, 5004 – 5010. Magnus, T., Chan, A., Savill, J., Tokya, K.V., Gold, R., 2002. Phagocytotic removal of apoptotic, inflammatory lymphocytes in the central nervous system by microglia and its functional implications. J. Neuroimmunol. 130, 1 – 9. Morioka, T., Baba, T., Black, K.L., Streit, W.J., 1992. Immunophenotypic analysis of infiltrating leukocytes and microglia in an experimental rat glioma. Acta Neuropathol. 83, 590 – 597. Parney, I.F., Hao, C.H., Petruk, K.C., 2000. Glioma immunology and immunotherapy. Neurosurgery 46, 778 – 791. Paul, D.B., Read, S.B., Kulprathipanja, N.V., Gomez, G.G., KleinschmidtDeMasters, B.K., Schiltz, P.M., Kruse, C.A., 2003. Gamma interferon transduced 9L gliosarcoma. Cytokine gene therapy and its relevance to cellular therapy with alloreactive cytotoxic T lymphocytes. J. NeuroOncol. 64, 89 – 99. Raza, S.M., Lang, F.F., Aggarwal, B.B., Fuller, G.N., Wildrick, D.M., Sawaya, R., 2002. Necrosis and glioblastoma: a friend or a foe? A review and a hypothesis. Neurosurgery 51, 2 – 12.
Read, S.B., Kulprathipanja, N.V., Gomez, G.G., Paul, D.B., Winston, K.R., Robbins, J.M., Kruse, C.A., 2003. Human alloreactive CTL interactions with gliomas and with those having upregulated HLA expression from exogenous IFN-gamma or IFN-gamma gene modification. J. Interferon Cytokine Res. 23, 379 – 393. Sawada, M., Suzumura, A., Hosoya, H., Marunouchi, T., Nagatsu, T., 1999. Interleukin-10 inhibits both production of cytokines and expression of cytokine receptors in microglia. J. Neurochem. 72, 1466 – 1471. Seguin, R., Biernacki, K., Prat, A., Wosik, K., Kim, H.J., Blain, M., McCrea, E., Bar-Or, A., Antel, J.P., 2003. Differential effects of Th1 and Th2 lymphocyte supernatants on human microglia. Glia 42, 36 – 45. Smith, M.E., 2001. Phagocytic properties of microglia in vitro: implications for a role in multiple sclerosis and EAE. Microsc. Res. Tech. 54, 81 – 94. Streit, W.J., Walter, S.A., Pennell, N.A., 1999. Reactive microgliosis. Prog. Neurobiol. 57, 563 – 581. Taniguchi, Y., Ono, K., Yoshida, S., Tanaka, R., 2000. Antigen-presenting capability of glial cells under glioma-harboring conditions and the effect of glioma-derived factors on antigen presentation. J. Neuroimmunol. 111, 177 – 185. Virasch, N., Kruse, C.A., 2001. Strategies using the immune system for therapy of brain tumors. Hematol. Oncol. Clin. North Am. 15, 1053 – 1071. Witting, A., Muller, P., Herrmann, A., Kettenmann, H., Nolte, C., 2000. Phagocytic clearance of apoptotic neurons by microglia/brain macrophages in vitro: involvement of lectin-, integrin-, and phosphatidylserine-mediated recognition. J. Neurochem. 75, 1060 – 1070.
Microglia phagocytose alloreactive CTL-damaged 9L gliosarcoma cells Nisha V. Kulprathipanja a, Carol A. Kruse a,b,* a
Department of Immunology, University of Colorado Health Sciences Center, 4200 E. 9th Avenue, B216, Denver, CO 80262, USA b Department of Pathology, University of Colorado Health Sciences Center, Room 2653 Campus Box B216, 4200 E. Ninth Avenue, Denver, CO 80262, USA Received 15 March 2004; received in revised form 21 April 2004; accepted 21 April 2004
Abstract Intracranial adoptive transfers of alloreactive cytotoxic T lymphocytes (aCTL) for brain tumor treatment were safe and showed promise in preclinical and early clinical trials. To better understand the endogenous immune responses that may ensue following cellular therapy with aCTL, we examined the ability of microglia to phagocytose aCTL-damaged and undamaged rat 9L gliosarcoma cells in vitro and in vivo. In vitro, 5.5 F 0.9% of microglial cells isolated from adult tumor-bearing rat brains phagocytosed aCTL-damaged 9L cells, whereas microglia did not bind to or ingest undamaged 9L cells. Addition of supernates from either 9L cell cultures or from aCTL + 9L co-incubate cell cultures to microglia did not significantly alter their ability to bind to or phagocytose damaged glioma cells even though the latter contained T helper 1 and 2 cytokines. At 3 days following intracranial 9L cell infusion, 17.5 F 0.1% of the microglia phagocytosed CFSE-labeled aCTL-damaged 9L tumor cells within the adult rat brain, confirming the in vitro data. The results suggest that microglia within the tumor microenvironment of the adult rat glioma model selectively remove damaged, but not undamaged, glioma cells. D 2004 Elsevier B.V. All rights reserved. Keywords: Microglia; Phagocytosis; Glioma; Antigen presentation; Apoptosis; CTL
1. Introduction The primary goal of therapies for malignant gliomas is to induce tumor cell death. However, the role(s) that microglia may play in the fate of the damaged glioma cells is an issue that has not been extensively investigated. Microglia have been observed in high numbers in human brain tumors (Graeber et al., 2002; Lorusso and Rossi, 1997) and in experimental rat glioma models (Badie and Schartner, 2000; Morioka et al., 1992). Because microglial cells derived from neonatal rodent and human brains phagocytose neuronal and myelin debris (Magnus et al., 2002; Smith, 2001; Streit et al., 1999; Witting et al., 2000), it is likely that microglial cells are involved in the clearance of damaged glioma cells within the brain. In vitro studies by Chang et al. (2000) support this idea by demonstrating the ability of human microglia to phagocytose apoptotic glioma cells. * Corresponding author. Present address: La Jolla Institute for Molecular Medicine, 4570 Executive Drive, Ste 100, San Diego, CA 92121, USA. Tel.: +1-858-587-8788x142; fax: +1-858-587-6742. E-mail address: [email protected] (C.A. Kruse). 0165-5728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2004.04.011
Our laboratory has been studying cellular immune therapies that have the potential to selectively destroy malignant brain tumor cells while leaving normal brain cells unharmed. We have shown that one specific type of effector cell population, alloreactive cytotoxic T lymphocytes (aCTL) sensitized to the major histocompatibility complex (MHC) antigens of the tumor host, was tolerable and showed promise when administered intracranially in preclinical and clinical studies (Fleshner et al., 1992; Kruse et al., 1997, 1990, 1994b; Kruse and Rubinstein, 2001; Read et al., 2003). Therefore, we are particularly interested in the interaction of other immune cells, such as microglia, with glioma cells that have been damaged by aCTL treatment. Information on whether microglial cells are capable of removing damaged gliomas undergoing apoptosis or necrosis is important for designing treatments that minimize damage to normal brain tissue and inflammatory processes in the brain. In this study, we investigated the ability of microglia/macrophages isolated from tumor-bearing adult rat brains to phagocytose undamaged, ultraviolet radiation (UV)-damaged, and aCTL-damaged 9L glioma cells in vitro and in vivo.
N.V. Kulprathipanja, C.A. Kruse / Journal of Neuroimmunology 153 (2004) 76–82
2. Materials and methods 2.1. Cell culture, antibodies, and reagents The rat 9L gliosarcoma cell line was maintained in RPMI-1640 and Dulbecco’s Modified Eagle’s Medium (DMEM) (2:1 vol/vol) supplemented with 10% fetal bovine serum (FBS), 200 U/ml penicillin, and 200 Ag/ml streptomycin (Abs). Microglia were maintained in DMEM with 10% FBS and Abs. aCTL were cultured in Iscove’s Modified Dulbecco’s Medium supplemented with 10% FBS, 2 mM L-glutamine, 50 AM 2-mercaptoethanol, 0.5 mM N-monomethyl-L-arginine-HOAc (L-NMMA, CBC Cyclopss Biochemical, Salt Lake City, UT), 50 Ag/ml gentamicin, and Abs. Cells were kept in 5% CO2 at 37 jC in a humidified incubator. Mouse anti-rat CD11bphycoerythrin (PE), mouse anti-rat CD11b-allophycocyanin (APC), mouse IgG2a-APC isotype control, and annexin-V-APC were purchased from Caltag Laboratories, Burlingame, CA. The mouse IgG2a-PE isotype control was purchased from Beckman Coulter, Fullerton, CA. Propidium iodide (PI) was from R&D Systems, Minneapolis, MN. Carboxy-fluorescein diacetate, succinimidyl ester (CFSE) was from Molecular Probes, Eugene, OR, and 7amino actinomycin D (7-AAD) was from ICN Biomedicals, Irvine, CA.
77
er lymphocytes were then mixed at a 1:1 ratio in a one-way mixed lymphocyte reaction, and the aCTL were used after 5 –7 days in culture. 2.5. Isolation of aCTL-damaged, UV-damaged, and undamaged 9L cells aCTL and 9L cells (pre-labeled with 0.5 AM CFSE according to manufacturer’s protocol) were co-incubated at an effector to target ratio of 10:1 for 18 –24 h at 37 jC in a 5% CO2 humidified chamber. The supernate was then collected and clarified (12,500 gmin) for use in the phagocytosis assays. The cells were harvested and labeled with 200 Ag/ml 7-AAD. After washing with PBS, a portion of the cells was stained with the phosphatidylserine (PS) early apoptosis marker, annexin-V-APC, and with PI. The aCTL-damaged glioma cells (CFSE and 7-AAD positive) were then sorted from the remaining cells using a flow cytometer (MoFlo Cell Sorter, DakoCytomation, Fort Collins, CO). Damaged 9L cells that had detached from the culture flask 30 h after a 10-min exposure to 40 AW/cm2 UV and untreated 9L cells were added as the controls in the phagocytosis assay. The UV-damaged and undamaged 9L cells were also labeled with CFSE prior to use in the assays, and their damage profiles were assessed using 7-AAD and annexin-V-APC/PI flow cytometric assays.
2.2. Intracranial infusion of tumor 2.6. In vitro phagocytosis assay 9L cells were intracranially implanted into anesthetized adult (6 –8 weeks) F344 rats as described before (Kruse et al., 1990). Two infusions of 9L cells (5 105 cells/10 Al) in phosphate buffered saline (PBS) were infused at a depth of 4 mm below the dura using a 50-Al Hamilton syringe through small burr holes at 3 mm to the right and to the left of the midline, 2 mm anterior to the bregma. Microglia were recovered from brain tissue 7– 10 days post-tumor infusion. 2.3. Isolation of microglia Microglia were isolated as described by Ford et al. (1995) with minor modifications. Briefly, brains from normal adult (6– 8 weeks) rats or rats bearing intracranial 9L tumors were minced with scissors and mechanically dissociated with a glass homogenizer. The microglia were collected using two discontinuous Percoll (Amersham Biosciences, Piscataway, NJ) gradients and were washed twice before use.
Microglia were added to 24-well plates at 2 105 cells/ well and were allowed to adhere to the plates for 2 h. CFSE-labeled aCTL-damaged, UV-damaged, or undamaged 9L glioma cells were added to the microglial monolayers for 3 h. In select wells, microglial cells were placed in 24-h supernate from either 9L + aCTL co-cultures or from undamaged 9L cultures during the phagocytosis assay. Cytochalasin D (5 Ag/ml final concentration, Sigma, St. Louis, MO), which prevents phagocytic function by inhibiting actin cytoskeleton polymerization, was added to one set of control wells containing microglia 30 min prior to the addition of 9L cells (Jersmann et al., 2003). The cells were harvested, stained with CD11b-APC, and fixed in 1% formaldehyde in PBS. Cells were analyzed using a flow cytometer (BD FACSCalibur, Becton Dickinson, San Jose, CA). Phagocytosis was evaluated by subtracting the background binding observed in the control wells from the percentage of CD11b+ microglia also positive for CFSE in the experimental wells.
2.4. Generation of aCTL 2.7. In vivo phagocytosis assay aCTL were prepared and isolated as described before (Paul et al., 2003). F344 rats (6– 8 weeks) were the source of stimulator lymphocytes and DA or Sprague – Dawley rats (6– 8 weeks) were the source of responder lymphocytes. Stimulator (60Co-gamma irradiated at 2500 rad) and respond-
aCTL-damaged, UV-damaged, and undamaged 9L glioma cells labeled with 10 AM CFSE were intracranially implanted into adult rat brains as described above. Control rats were infused with aCTL-damaged, UV-damaged, or
78
N.V. Kulprathipanja, C.A. Kruse / Journal of Neuroimmunology 153 (2004) 76–82
Fig. 1. Flow cytometric analysis (left panel) of microglia from normal adult rat brains stained with CD11b-PE (open) and an isotype antibody (shaded). Microglia from tumor-bearing adult rat brains 2 h after plating as observed by light microscopy (right panel, bar = 10 Am).
undamaged 9L cells that were not CFSE labeled. Microglial cells were isolated from the rat brains at 3 or 5 days after glioma infusion and were stained with CD11b prior to analysis by flow cytometry. The gates for analyzing phagocytic microglial cells (CFSE positive) were set using the population of microglial cells from the control rats (CFSE negative).
3. Results 3.1. Microglial cell yields are higher from glioma-bearing brains than from normal adult rat brains Microglia freshly isolated from normal (Fig. 1, left panel) and 9L tumor-bearing adult rat brains expressed
CD11b on their cell surfaces, and the cells adhering to the plating surface primarily displayed an amoeboid morphology (Fig. 1, right panel). Approximately two-thirds of the CD11b+ cells were of the CD45low microglial phenotype (data not shown), implicating the remaining CD11b+ cells as CNS macrophages (Ford et al., 1995). For our assays, all CD11b+ cells were used. The microglial cell yields from intracranial tumor-bearing rat brains were approximately 10-fold higher, at 14.6 104 to 37.8 104 cells/rat (average 29.5 104), than from normal brains which ranged from 1.4 104 to 5.4 104 cells/brain (average 2.7 104). Cell viabilities of freshly isolated microglia, as assessed by trypan blue dye exclusion, were 70% to 95% (n = 15). Similar microglial cell yields (data not shown) were obtained from rats bearing CNS-1 gliomas (Kruse et al., 1994c). 3.2. 9L cells are injured upon exposure to aCTL or UV radiation 9L cell injury was confirmed by flow cytometric analysis of 7-AAD and annexin-V/PI staining. The majority (87 – 94%) of untreated 9L cells were viable (Fig. 2A and D). However, 96% of UV-exposed and 66% of aCTLexposed 9L cells incorporated 7-AAD into their DNA, reflecting a high percentage of injured cells in both populations (Fig. 2B and C). Moreover, 48% of both the UV-exposed and aCTL-exposed cells expressed the early apoptotic marker, annexin-V (Fig. 2E and F). 9L cells damaged by UV and aCTL exposure (Fig. 3C and D) also
Fig. 2. 9L glioma cell injury assessed by 7-AAD (top panels) and annexin-V-APC/PI (bottom panels) flow cytometric assays. Flow diagrams are shown for untreated (A, D), UV-exposed (B, E), and aCTL-exposed (C, F) 9L cells.
N.V. Kulprathipanja, C.A. Kruse / Journal of Neuroimmunology 153 (2004) 76–82
79
Fig. 3. Laser scatter profile of (A) microglial cells, (B) microglia and undamaged 9L cells, (C) microglia and UV-damaged 9L cells, and (D) microglia and aCTL-damaged 9L cells from an in vitro phagocytosis assay. The microglial cells, which are encircled by the oval gate in the dot-plot, were confirmed to express CD11b.
3.3. Microglial cells bind to and phagocytose UV-damaged and aCTL-damaged 9L, but not undamaged 9L cells in vitro
nates from 9L cell or aCTL + 9L cell cultures did not significantly alter the ability of the microglia to bind to or phagocytose glioma cells (data not shown), even though multiple cytokines (IFN-gamma, TNF-alpha, GM-CSF, IL6, and IL-10) were detected in the aCTL + 9L cell culture supernate, but not the 9L culture supernates (data not shown).
Microglial cells in the phagocytosis assays were easily identified by flow cytometry in forward scatter/side scatter dot-plots (Fig. 3, oval gates), and 60– 80% of the cells within the oval gates expressed CD11b (data not shown). The microglial cells displaying CD11b were then analyzed for CFSE positivity to assess the ability of microglia to bind/phagocytose CFSE-labeled 9L cells. The background binding was observed by blocking phagocytosis with cytochalasin D. 6.8 F 1.7% and 4.3 F 0.9% of the microglia bound to UV-damaged and aCTL-damaged 9L cells, respectively, but did not bind to undamaged 9L cells after a 3-h co-incubation (Fig. 4, black bars). Phagocytosis percentages were then calculated by subtracting the binding control percentages from the percentages of cells not exposed to cytochalasin D (Fig. 4, white bars). The microglia phagocytosed UV-damaged (8.1 F 0.4%) and aCTL-damaged (5.5 F 0.9%) 9L cells, but did not phagocytose undamaged 9L cells. Moreover, addition of super-
Fig. 4. The microglial cells positive for CD11b (oval gates, Fig. 3) were analyzed for CFSE positivity to indicate binding (cytochalasin D+) and binding plus phagocytic uptake (cytochalasin D-) of CFSE-labeled 9L cells in vitro. Microglia bind to (black bars) and phagocytose (white bars minus black bars) UV-damaged and aCTL-damaged, but not undamaged 9L glioma cells. Binding and phagocytosis from three independent experiments given as a percentage of CFSE-positive cells F S.E.M.
displayed decreased forward scatter profiles as compared to undamaged 9L cells (Fig. 3B), confirming the smaller cell sizes resulting from cell injury processes (Darzynkiewicz et al., 1997).
80
N.V. Kulprathipanja, C.A. Kruse / Journal of Neuroimmunology 153 (2004) 76–82
Fig. 5. In vivo microglial phagocytosis of undamaged, UV-damaged, and aCTL-damaged 9L glioma cells at days 3 and 5 after intracranial implantation. Phagocytosis from two independent experiments (2 rats/ group) given as a percentage of CFSE-positive cells F S.E.M.
3.4. Microglial cells phagocytose UV-damaged 9L and aCTL-damaged 9L, but not undamaged 9L cells in vivo Microglial cells were isolated and analyzed 3 and 5 days after intracranial implantation of either CFSE-labeled undamaged, UV-damaged, or aCTL-damaged 9L cells into adult rat brains (Fig. 5). Undamaged 9L cells were not phagocytosed by microglia in vivo at either 3 or 5 days. However, at 3-days post-implantation, 9.7 F 4.4% and 17.5 F 0.1% of the microglia phagocytosed CFSE-labeled UV-damaged and aCTL-damaged 9L cells, respectively. Few microglial cells were CFSE positive at 5 days, suggesting that the microglia degraded the ingested 9L cells by that time point.
4. Discussion Although cultured microglia from neonatal rat/mice brains are widely used in in vitro experiments, they are more activated than the resting, ramified microglia residing in adult rat brains (Becher and Antel, 1996; Carson et al., 1998, 1999). Therefore, microglial cells used in this study were isolated from adult tumor-bearing rat brains to more accurately reflect the activation state and phagocytic function of microglia present clinically in glioma patients. In addition, microglia were extracted from rat brains with an established tumor to better resemble the microglia found within glioma patients since the cells were likely exposed to a milieu of cytokines and chemokines within the tumor microenvironment, including several immunosuppressive factors (Fakhrai et al., 1996; Gillespie, 1996; Hao et al., 2002; Kiefer et al., 1994; Parney et al., 2000; Kielian et al., 2002; Read et al., 2003; Virasch and Kruse, 2001). As we and others (Badie and Schartner, 2000; Graeber et al., 2002; Lorusso and Rossi, 1997; Morioka et al., 1992) demonstrated, microglia are ubiquitous in glioma-bearing tissue. They are in close proximity to both healthy tumor cells and necrotic tumor cells, the latter being a feature of glioblastoma multiforme (Raza et al., 2002). Moreover, the
number of damaged glioma cells in juxtaposition with microglia increases after the administration of cytoreductive therapies that induce cell damage through apoptotic and necrotic pathways (Finkel, 1999; Gomez et al., 2004; Henkart et al., 1997; Jerome et al., 2003; Read et al., 2003). In particular, aCTL migrate from the instillation site through brain parenchyma (Kruse et al., 1994a), which increases the likelihood of tumor/effector cell contact, subsequent glioma cell damage, and microglial recognition of the damaged glioma cells. We have extensively studied intracranial adoptive transfer of aCTL into the brain preclinically and clinically (Fleshner et al., 1992; Kruse et al., 1997, 1990, 1994b; Kruse and Rubinstein, 2001). The responses noted may be a combined result of (1) direct interactions of the ex vivoactivated CTL effectors with the tumor cells, (2) cytokines secreted by the ex vivo-introduced effector CTL, (3) cytokines/chemokines produced by the glioma cells, and (4) interactions of endogenous immune cell components, including infiltrating microglial and T cells, with aCTL and glioma cells. Thus, analysis of microglial cell roles in aCTL cellular therapy is essential. The activation state of microglia after their interactions with damaged glioma cells, endogenous lymphocytes, adoptive therapeutic cells, and exposure to cytokines may have contradictory implications for the ability of the immune system to eliminate brain tumors (Badie and Schartner, 2001). Microglial immune functions are stimulated by pro-inflammatory T-helper 1 cytokines (Grau et al., 1997; Seguin et al., 2003), but are suppressed by anti-inflammatory T-helper 2 cytokines (Sawada et al., 1999). Phagocytosis of apoptotic cells has been shown to result in antiinflammatory responses (Hirt and Leist, 2003; Magnus et al., 2001; Witting et al., 2000), whereas exposure to necrotic debris results in pro-inflammatory responses (Fadok et al., 2001). Therefore, microglial phagocytosis of apoptotic gliomas may contribute to the existing array of immunoinhibitory factors within the tumor, which are also crucial for maintaining control over inflammation in the confined space of the brain. On the other hand, microglial uptake of damaged glioma cells may stimulate their secretion of proinflammatory signals, thereby maintaining microglia in an activated state. The activation state of microglia is crucial to their ability to present antigen to lymphocytes. We (unpublished data) and others (Badie and Schartner, 2000) demonstrated that microglia from tumor-bearing brains express major histocompatibility complex (MHC) class II and B7 costimulatory molecules that are necessary for antigen presentation. However, studies indicate the poor ability of neonatal microglia to present tumor antigen to T cells (Flugel et al., 1999; Taniguchi et al., 2000). The microenvironment surrounding aCTL-treated gliomas would likely contain high levels of pro-inflammatory cytokines and chemokines (IFN-gamma, TNF-alpha, and GM-CSF) since they were observed in supernates from rat and human aCTL/glioma cell co-incu-
N.V. Kulprathipanja, C.A. Kruse / Journal of Neuroimmunology 153 (2004) 76–82
bates (Read et al., 2003 and unpublished data). These cytokines may help maintain the activation state of microglial cells and increase their ability to present glioma antigens to T cells. In summary, we have confirmed that brains bearing tumors have higher infiltrates of microglia, making an assessment of their role there important. We confirm that aCTL treatment injured glioma cells and that the injury was sufficient for a small percentage of the microglial cells to recognize, bind to, and phagocytose the glioma cells both in vitro and in vivo. Microglial cell exposure to and uptake of injured glioma cells is likely an essential precursor to tumor antigen presentation. Therefore, microglia may play an important role in effecting the overall response observed with aCTL adoptive immunotherapy. Since activated microglia display some MHC Class I (Grau et al., 1997), they theoretically may be subject to aCTL damage. If the role of microglia as antigen-presenting cells is important in the overall therapy, their injury is not particularly worrisome, since they could be adoptively transferred into the brain subsequent to aCTL therapy. Further exploration in this model to assess the ability of adult microglial cells to present glioma cell antigen upon treatment with aCTL is warranted.
Acknowledgements This work was supported in part by National Institutes of Health (NIH) Grants RO1-NS56798 to CAK, and F31 NS 44074 to NVK and the R. Herbert and Alma S. Manweiler Memorial Research Fund.
References Badie, B., Schartner, J.M., 2000. Flow cytometric characterization of tumorassociated macrophages in experimental gliomas. Neurosurgery 46, 957 – 961. Badie, B., Schartner, J., 2001. Role of microglia in glioma biology. Microsc. Res. Tech. 54, 106 – 113. Becher, B., Antel, J.P., 1996. Comparison of phenotypic and functional properties of immediately ex vivo and cultured human adult microglia. Glia 18, 1 – 10. Carson, M.J., Reilly, C.R., Sutcliffe, J.G., Lo, D., 1998. Mature microglia resemble immature antigen-presenting cells. Glia 22, 72 – 85. Carson, M.J., Sutcliffe, J.G., Campbell, I.L., 1999. Microglia stimulate naive T-cell differentiation without stimulating T-cell proliferation. J. Neurosci. Res. 55, 127 – 134. Chang, G.H.F., Barbaro, N.M., Pieper, R.O., 2000. Phosphatidylserinedependent phagocytosis of apoptotic glioma cells by normal human microglia, astrocytes, and glioma cells. Neuro-Oncology 2, 174 – 183. Darzynkiewicz, Z., Juan, G., Li, X., Gorczyca, W., Murakami, T., Traganos, F., 1997. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 27, 1 – 20. Fadok, V.A., Bratton, D.L., Guthrie, L., Henson, P.M., 2001. Differential effects of apoptotic versus lysed cells on macrophage production of cytokines: role of proteases. J. Immunol. 166, 6847 – 6854. Fakhrai, H., Dorigo, O., Shawler, D.L., Lin, H., Mercola, D., Black, K.L., Royston, I., Sobol, R.E., 1996. Eradication of established intracranial
81
rat gliomas by transforming growth factor beta antisense gene therapy. Proc. Natl. Acad. Sci. U. S. A. 93, 2909 – 2914. Finkel, E., 1999. Biomedicine—does cancer therapy trigger cell suicide? Science 286, 2256 – 2258. Fleshner, M., Watkins, L.R., Redd, J.M., Kruse, C.A., Bellgrau, D., 1992. A 9L gliosarcoma transplantation model for studying adoptive immunotherapy into the brains of conscious rats. Cell Transplant 1, 307 – 312. Flugel, A., Labeur, M.S., Grasbon-Frodl, E.M., Kreutzberg, G.W., Graeber, M.B., 1999. Microglia only weakly present glioma antigen to cytotoxic T cells. Int. J. Dev. Neurosci. 17, 547 – 556. Ford, A.L., Goodsall, A.L., Hickey, W.F., Sedgwick, J.D., 1995. Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared. J. Immunol. 154, 4309 – 4321. Gillespie, G.Y., 1996. Cytokines as modulators of malignant glioma progression. In: Ransohoff, R.M., Benveniste, E.N. (Eds.), Cytokines and the CNS. CRC Press, Boca Raton, pp. 269 – 286. Gomez, G.G., Read, S.B., Gerschenson, L.E., Santoli, D., Zweifach, A., Kruse, C.A., 2004. Interactions of the allogeneic effector leukemic T cell line, TALL-104, with human malignant brain tumors. Neuro-Oncology 6, 83 – 95. Available at: http://neuro-oncology.mc.duke.edu. Graeber, M.B., Scheithauer, B.W., Kreutzberg, G.W., 2002. Microglia in brain tumors. Glia 40, 252 – 259. Grau, V., Herbst, B., VanderMeide, P.H., Steiniger, B., 1997. Activation of microglial and endothelial cells in the rat brain after treatment with interferon-gamma in vivo. Glia 19, 181 – 189. Hao, C.H., Parney, I.F., Roa, W.H., Turner, J., Petruk, K.C., Ramsay, D.A., 2002. Cytokine and cytokine receptor mRNA expression in human glioblastomas: evidence of Th1, Th2 and Th3 cytokine dysregulation. Acta Neuropathol. 103, 171 – 178. Henkart, P.A., Williams, M.S., Zacharchuk, C.M., Sarin, A., 1997. Do CTL kill target cells by inducing apoptosis? Semin. Immunol. 9, 135 – 144. Hirt, U.A., Leist, M., 2003. Rapid, noninflammatory and PS-dependent phagocytic clearance of necrotic cells. Cell Death Differ. 10, 1156 – 1164. Jerome, K.R., Sloan, D.D., Aubert, M., 2003. Measurement of CTL-induced cytotoxicity: the caspase 3 assay. Apoptosis 8, 563 – 571. Jersmann, H.P.A., Ross, K.A., Vivers, S., Brown, S.B., Haslett, C., Dransfield, I., 2003. Phagocytosis of apoptotic cells by human macrophages: analysis by multiparameter flow cytometry. Cytometry Part A 51A, 7 – 15. Kiefer, R., Supler, M.L., Toyka, K.V., Streit, W.J., 1994. In-situ detection of transforming growth-factor-beta messenger-RNA in experimental rat glioma and reactive glial-cells. Neurosci. Lett. 166, 161 – 164. Kielian, T., van Rooijen, N., Hickey, W.F., 2002. MCP-1 expression in CNS-1 astrocytoma cells: implications for macrophage infiltration into tumors in vivo. J. Neuro-Oncol. 56, 1 – 12. Kruse, C.A., Rubinstein, D., 2001. Cytotoxic T-lymphocytes reactive to patient major histocompatibility complex proteins for therapy of brain tumors. In: Liau, L.M., Becker, D.P., Cloughesy, T.F., Bigner, D.D. (Eds.), Brain Tumor Immunotherapy. Humana Press, Totowa, NJ, pp. 149 – 170. Kruse, C.A., Lillehei, K.O., Mitchell, D.H., Kleinschmidt-DeMasters, B., Bellgrau, D., 1990. Analysis of Interleukin-2 and various effector cellpopulations in adoptive immunotherapy of 9L rat gliosarcoma-allogeneic cytotoxic T-lymphocytes prevent tumor take. Proc. Natl. Acad. Sci. U. S. A. 87, 9577 – 9581. Kruse, C.A., Kong, Q.Z., Schiltz, P.M., Kleinschmidt-DeMasters, B.K., 1994a. Migration of activated lymphocytes when adoptively transferred into cannulated rat-brain. J. Neuroimmunol. 55, 11 – 21. Kruse, C.A., Schiltz, P.M., Bellgrau, D., Kong, Q.Z., Kleinschmidt-DeMasters, B.K., 1994b. Intracranial administrations of single or multiple source allogeneic cytotoxic T-lymphocytes. Chronic therapy for primary brain-tumors. J. Neuro-Oncol. 19, 161 – 168. Kruse, C.A., Molleston, M.C., Parks, E.P., Schiltz, P.M., KleinschmidtDeMasters, B.K., Hickey, W.F., 1994c. A rat glioma model, CNS-1
82
N.V. Kulprathipanja, C.A. Kruse / Journal of Neuroimmunology 153 (2004) 76–82
with invasive characteristics similar to those of human gliomas—a comparison to 9L gliosarcoma. J. Neuro-Oncol. 22, 191 – 200. Kruse, C.A., Cepeda, L., Owens, B., Johnson, S.D., Stears, J., Lillehei, K.O., 1997. Treatment of recurrent glioma with intracavitary alloreactive cytotoxic T lymphocytes and interleukin-2. Cancer Immunol. Immunother. 45, 77 – 87. Lorusso, L., Rossi, M.L., 1997. The phagocyte in human gliomas. Ann. N. Y. Acad. Sci. 832, 405 – 425. Magnus, T., Chan, A., Grauer, O., Toyka, K.V., Gold, R., 2001. Microglial phagocytosis of apoptotic inflammatory T cells leads to down-regulation of microglial immune activation. J. Immunol. 167, 5004 – 5010. Magnus, T., Chan, A., Savill, J., Tokya, K.V., Gold, R., 2002. Phagocytotic removal of apoptotic, inflammatory lymphocytes in the central nervous system by microglia and its functional implications. J. Neuroimmunol. 130, 1 – 9. Morioka, T., Baba, T., Black, K.L., Streit, W.J., 1992. Immunophenotypic analysis of infiltrating leukocytes and microglia in an experimental rat glioma. Acta Neuropathol. 83, 590 – 597. Parney, I.F., Hao, C.H., Petruk, K.C., 2000. Glioma immunology and immunotherapy. Neurosurgery 46, 778 – 791. Paul, D.B., Read, S.B., Kulprathipanja, N.V., Gomez, G.G., KleinschmidtDeMasters, B.K., Schiltz, P.M., Kruse, C.A., 2003. Gamma interferon transduced 9L gliosarcoma. Cytokine gene therapy and its relevance to cellular therapy with alloreactive cytotoxic T lymphocytes. J. NeuroOncol. 64, 89 – 99. Raza, S.M., Lang, F.F., Aggarwal, B.B., Fuller, G.N., Wildrick, D.M., Sawaya, R., 2002. Necrosis and glioblastoma: a friend or a foe? A review and a hypothesis. Neurosurgery 51, 2 – 12.
Read, S.B., Kulprathipanja, N.V., Gomez, G.G., Paul, D.B., Winston, K.R., Robbins, J.M., Kruse, C.A., 2003. Human alloreactive CTL interactions with gliomas and with those having upregulated HLA expression from exogenous IFN-gamma or IFN-gamma gene modification. J. Interferon Cytokine Res. 23, 379 – 393. Sawada, M., Suzumura, A., Hosoya, H., Marunouchi, T., Nagatsu, T., 1999. Interleukin-10 inhibits both production of cytokines and expression of cytokine receptors in microglia. J. Neurochem. 72, 1466 – 1471. Seguin, R., Biernacki, K., Prat, A., Wosik, K., Kim, H.J., Blain, M., McCrea, E., Bar-Or, A., Antel, J.P., 2003. Differential effects of Th1 and Th2 lymphocyte supernatants on human microglia. Glia 42, 36 – 45. Smith, M.E., 2001. Phagocytic properties of microglia in vitro: implications for a role in multiple sclerosis and EAE. Microsc. Res. Tech. 54, 81 – 94. Streit, W.J., Walter, S.A., Pennell, N.A., 1999. Reactive microgliosis. Prog. Neurobiol. 57, 563 – 581. Taniguchi, Y., Ono, K., Yoshida, S., Tanaka, R., 2000. Antigen-presenting capability of glial cells under glioma-harboring conditions and the effect of glioma-derived factors on antigen presentation. J. Neuroimmunol. 111, 177 – 185. Virasch, N., Kruse, C.A., 2001. Strategies using the immune system for therapy of brain tumors. Hematol. Oncol. Clin. North Am. 15, 1053 – 1071. Witting, A., Muller, P., Herrmann, A., Kettenmann, H., Nolte, C., 2000. Phagocytic clearance of apoptotic neurons by microglia/brain macrophages in vitro: involvement of lectin-, integrin-, and phosphatidylserine-mediated recognition. J. Neurochem. 75, 1060 – 1070.