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Effects of polyclonal immunoglobulins and other immunomodulatory agents on microglial phagocytosis of apoptotic inflammatory T-cells
Journal of Neuroimmunology 135 (2003) 161 – 165 www.elsevier.com/locate/jneuroim
Short communication
Effects of polyclonal immunoglobulins and other immunomodulatory agents on microglial phagocytosis of apoptotic inflammatory T-cells Andrew Chan *,1, Christina Papadimitriou 1,2, Wolfgang Graf, Klaus V. Toyka, Ralf Gold Department of Neurology, Clinical Research Group for Multiple Sclerosis and Neuroimmunology, Julius-Maximilians-University, D-97080 Wu¨rzburg, Germany Received 3 September 2002; received in revised form 25 November 2002; accepted 25 November 2002
Abstract T-cell apoptosis in the CNS is an effective mechanism for the noninflammatory resolution of autoimmune T-cell infiltrates. Ingestion of apoptotic leukocytes by microglia results in an efficient clearance of the inflammatory infiltrate, followed by a profound downregulation of proinflammatory phagocyte immune functions. The effects of different immunomodulatory agents on Lewis rat microglial phagocytosis of apoptotic autologous thymocytes or myelin-basic protein (MBP)-specific, encephalitogenic T-cells were investigated using a standardized, light microscopical in vitro phagocytosis assay. Pretreatment of microglia with polyclonal 7S immunoglobulins (IVIg) decreased the phagocytosis of apoptotic thymocytes by 38.2% ( p < 0.0001). Also, immunoglobulin F(abV)2 fragments decreased microglial phagocytosis, suggesting an Fc receptor-independent mechanism. Similar results were obtained using MBP-specific T-cells. Pretreatment of microglia with IFN-g increased the phagocytosis of apoptotic cells by 65.4%, which was to a large extent counteracted by IVIg. Glatiramer acetate (GLAT) did not exert an effect on microglial phagocytosis, while methylprednisolone (MP) induced microglial apoptosis in vitro. These results indicate that IVIg has a high potential to inhibit microglial phagocytosis of apoptotic inflammatory T-cells even under proinflammatory conditions and extend our view of the complex immunomodulatory effects of IVIg. D 2002 Elsevier Science B.V. All rights reserved. Keywords: T-cell apoptosis; Multiple sclerosis; Experimental autoimmune encephalomyelitis; Glatiramer acetate; Glucocorticosteroids
1. Introduction Apoptosis of 30 –50% of all invading T-cells represents a crucial mechanism in the termination of autoimmune T-cellmediated inflammation in the human and rodent CNS, contributing to clinical recovery in experimental autoimmune encephalomyelitis (EAE) and acute disseminated leukoencephalomyelitis in man (ADEM) (Bauer et al., 2001; Pender and Rist, 2001). A key event in the resolution of an inflammatory infiltrate is the nonphlogistic and thus safe phagocytic clearance of
* Corresponding author. Neurologische Universita¨tsklinik, JosefSchneider-Straße 11, D-97080 Wu¨rzburg, Germany. Tel.: +49-931-20124621; fax: +49-931-201-23488. E-mail address: [email protected] (A. Chan). 1 Equally contributing authors. 2 Present address: Department of Neurology, University Clinic, Ahepa Hospital, Thessaloniki, Greece.
apoptotic leukocytes by tissue-specific phagocytes (Fadok et al., 2001). Phagocytosis of apoptotic lymphocytes by macrophages/microglia, oligodendrocytes and astrocytes has been described in situ in Lewis rat EAE (Nguyen and Pender, 1998). Lewis rat microglia efficiently phagocytoses apoptotic, encephalitogenic MBP-specific T-cells in vitro, differentially regulated by Th1-/Th2-type cytokines (Chan et al., 2001). The phagocytosis of apoptotic T-cells by Lewis rat microglia is more efficient than by astrocytes and leads to a profound downregulation of microglial immune functions, making this process an attractive target for therapeutic interventions (Magnus et al., 2002). The immunomodulatory agent interferon-beta augments phagocytosis specifically of apoptotic inflammatory cells, and also adult human microglia obtained from normal brain tissue phagocytoses apoptotic inflammatory cells in vitro (Chan et al., 2002). Here we set out to investigate the effects of other therapeutically used immunomodulatory agents on Lewis rat microglial phagocytosis of apoptotic inflammatory cells.
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2. Materials and methods 2.1. Cell culture All cell culture media and supplements were obtained from Gibco/BRL (Eggenstein, Germany) unless otherwise noted. Microglial cells from neonatal Lewis rats (P0 –P2, Charles River, Sulzfeld, Germany) with a purity of consistently >97% were isolated as described before (Chan et al., 2001). Apoptosis of autologous thymocytes and of the MBPspecific, encephalitogenic CD4+ T-cell line MBP13 was induced by methylprednisolone (MP, Aventis Pharma, Bad Soden, Germany) as described in detail before (Chan et al., 2001). MP-treated thymocytes had a proportion of 37.8 F 2.6% (mean F S.E.M.) annexin V single-positive (early apoptotic) and 5.6 F 0.6% annexin V/PI-positive (late apoptotic/necrotic) cells, untreated control thymocytes were 12 F 2.6% annexin V-positive and 3.2 F 0.5% annexin V/PIpositive (Chan et al., 2001). The proportion of viable thymocytes or MBP13 cells excluding trypan blue was consistently >97%. 2.2. In vitro phagocytosis assay The standardized, microscopically quantified microglial phagocytosis assay has been described and illustrated in detail before (Chan et al., 2001). Four hundred microliters of a 0.75 106/ml suspension of microglial cells per well was seeded in 48-well plates (Costar) and cultured overnight at 37 jC/5% CO2 (BME, 10% FCS (Sigma Aldrich Chemie, Steinheim, Germany), 50 U/ml penicillin, 50 Ag/ ml streptomycin). Triplicate wells of microglial cells were then incubated with the respective concentrations of IVIg (SandoglobulinR, Novartis Pharma, Nu¨rnberg, Germany), F(abV)2 fragments (GammaveninR, Aventis Behring, Marburg, Germany), glatiramer acetate (GLAT, TEVA Pharma/ Aventis Pharma), MP or BME for 20– 24 h. Immunoglobulin preparations were dialyzed (H2O or BME) to remove stabilising agents that are potentially toxic in cell culture. Human albumin (Octapharma, Langenfeld, Germany; DRK Blutspendedienst, Baden-Baden, Germany) or ovalbumin (Sigma) were used as control proteins, respectively. In case of combined IFN-g (30 U/ml, R&D, Minneapolis, MN, USA))/IVIg pretreatment, microglia was either preincubated simultaneously or in another set of experiments sequentially treated with IFN-g (8 h) followed by IVIg (12 h). For RGDS/RGES peptide inhibition experiments, target cells were preincubated with the peptides (1– 2 mM, BME, 15 min, 4 jC) and subsequently added to the microglia without further washing. Thymocytes (500 Al, 20 106/ml in BME) or MBP13 T-cells (500 Al, 10 106/ ml) were co-cultured with the microglia (2 h, 37 jC, 5% CO2) followed by vigorous washing with cold PBS (4 jC) (Chan et al., 2001). After trypsinization, a separate cytocentrifuge preparation was obtained for each well and stained with May-Giemsa (Merck, Darmstadt, Germany)
(Chan et al., 2001). An average of 500 microglial cells per slide were counted in a blinded fashion by light microscopy. In some experiments, data is additionally given as phagocytic index (percent of phagocytosing microglia multiplied with the average number of ingested target cells per microglia) (Chan et al., 2001). All values are expressed as mean F S.E.M. Statistical significance was evaluated using Student’s t-test (GraphPad Software, San Diego, USA).
3. Results 3.1. IVIg and F(abV)2 fragments decrease microglial phagocytosis of apoptotic inflammatory cells As reported before, microglia has a high capacity to phagocytose apoptotic thymocytes or CNS autoantigenspecific, encephalitogenic T-cells in contrast to non-apoptotic target cells (Chan et al., 2001) (Figs. 1A and 2). As illustrated in Figs. 1A,B and 2, IVIg pretreatment (20 mg/ ml) decreased the phagocytosis of apoptotic thymocytes by 38.2 F 5.2% (mean F S.E.M.) in comparison to untreated microglia ( p < 0.0001). Pretreatment with human albumin (HA) did not exert an effect (Fig. 2). The decrease of phagocytosis was dose-dependent and reached a plateau with 20 mg/ml IVIg (percent inhibition, 10 mg/ ml: 26.3 F 6.25, p < 0.01; 30 mg/ml: 39.5 F 7.2%, p < 0.001). IVIg not only decreased the phagocytosis of corticosteroid-treated, apoptotic target cells, but also the much lower baseline phagocytosis of nontreated thymocytes, albeit to a lesser extent (Fig. 2, 27.5 F 3.4%, p < 0.0001). The stronger inhibitory IVIg effect on phagocytosis of apoptotic thymocytes was even more pronounced in the phagocytic index, which reflects the phagocytic capacity of individual microglial cells (IVIg 20 mg/ml, percent inhibition for apoptotic thymocytes: 32.6 F 7.5%, p < 0.001; for non-corticosteroid-treated cells: 18.2 F 6%, p < 0.05). IVIg (20 mg/ml) also decreased the phagocytosis of corticosteroid-treated, apoptotic, encephalitogenic MBP-specific MBP13 T-cells (percent inhibition 20.7 F 14.7%) and of non-corticosteroid-treated T-cells (13.6 F 5.6%). To investigate which portion of IVIg mediated the inhibition of phagocytosis, microglia was pretreated using F(abV)2 immunoglobulin fragments. Again, F(abV)2-mediated inhibition was more pronounced for the uptake of apoptotic thymocytes (49.1 F 3%, p < 0.0001) than for non-corticosteroid-treated cells (17.8 F 1.6%, p < 0.001). F(abV)2 fragments also decreased microglial phagocytosis of apoptotic MBP13 T-cells by 48.3 F 7% ( p < 0.0001), whereas no clear inhibition could be observed for non-glucocorticosteroidtreated T-cells. These results indicated that the suppression of phagocytosis of apoptotic and non-apoptotic cells was at least to a great part independent of Fc-receptor-mediated mechanisms.
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Fig. 2. Phagocytosis of autologous thymocytes by untreated and IVIgpretreated microglia. Microglia had a higher capacity for the uptake of corticosteroid-treated, apoptotic (+) thymocytes than for non-corticosteroidtreated ( ) thymocytes. IVIg decreased phagocytosis of apoptotic target cells and to a lesser extent of non-corticosteroid-treated cells in comparison to the untreated microglia, while human albumin (HA) did not have an effect on phagocytosis. Phagocytosis rate: given as percentage of the mean of the untreated controls (+) + S.E.M. 42.1 F 4.7% (mean F S.E.M.) of the untreated microglia were capable of phagocytosing apoptotic thymocytes. Five independent experiments, each performed in triplicates.
the phagocytic index by 88.4 F 29% ( p < 0.05), this effect was reduced by IVIg (20 mg/ml) by 69.4 F 2% ( p < 0.05). 3.3. Glatiramer acetate (GLAT) does not have an effect on microglial phagocytosis while methylprednisolone (MP) induces microglial apoptosis in vitro Pretreatment with GLAT (10 – 50 Ag/ml) did not show any specific effects on microglial phagocytosis of apoptotic or Fig. 1. Photomicrographs of untreated (A) or IVIg-pretreated (20 mg/ml) Lewis rat microglia (B) after 2 h interaction with autologous apoptotic thymocytes, which show typical apoptotic morphology with condensed chromatin (arrows). May-Giemsa stain. Bar = 10 Am.
3.2. IVIg partially counteracts the phagocytosis-promoting effect of interferon-gamma (IFN-c) As reported before, IFN-g increases microglial phagocytosis of apoptotic thymocytes, whereas the uptake of nonapoptotic target cells is not altered (Chan et al., 2001). IFN-g (30 U/ml) augmented the microglial phagocytosis rate for apoptotic thymocytes by 65.4 F 18.5% above the untreated controls (Fig. 3, p < 0.01), similar to values reported previously (Chan et al., 2001). Simultaneous preincubation of microglia with IFN-g and IVIg partially reversed this effect in an IVIg dose-dependent manner (percent inhibition of the IFN-g-augmented phagocytosis rate, IVIg 10 mg/ml: 33.2 F 8.4%; IVIg 20 mg/ml: 49.4 F 2.8%, p < 0.05). No change was observed with the combination of IFN-g and HA (Fig. 3). The inhibitory effect was even more pronounced using the phagocytic index. Whereas IFN-g alone increased
Fig. 3. Microglial phagocytosis of apoptotic thymocytes after pretreatment with IFN-g, IFN-g/IVIg or IFN-g/human albumin. IFN-g (30 IU/ml) increased the phagocytosis rate for apoptotic target cells (+), which was partially counteracted by the combination with IVIg (IFN-g/IVIg, 20 mg/ml). Combined pretreatment with IFN-g and human albumin (IFN-g/HA, 20 mg/ ml) did not alter the phagocytosis-promoting effect of IFN-g. Phagocytosis rate: given as percentage of the mean of the untreated controls (+) + S.E.M. 34.9 F 5.9% (mean F S.E.M.) of the untreated microglia were capable of phagocytosing apoptotic thymocytes. Two independent experiments, each performed in triplicates.
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non-corticosteroid-treated thymocytes in comparison to ovalbumine (data not shown). Pretreatment of microglia with MP (50 –1000 nM) resulted in decreased phagocytosis of thymocytes. Immunohistochemistry demonstrated a profound induction of microglial apoptosis even by low concentrations of MP with all cells showing annexin V/PI positivity after 3 h of MP treatment (200 nM, data not shown).
4. Discussion Although high dose IVIg are widely used in neurological diseases of presumed autoimmune etiology, their pleiotropic mechanisms of action are only incompletely understood (Kazatchkine and Kaveri, 2001; Wiles et al., 2002). More recently, a possible influence of IVIg also on local CNS immune reactions and remyelination has been suggested (Stangel et al., 2000a; Stangel and Compston, 2001, Warrington et al., 2000). Here, we have demonstrated that IVIg in concentrations corresponding to serum levels during IVIg treatment inhibit microglial phagocytosis of apoptotic thymocytes as well as MBP-specific, encephalitogenic T-cells. This inhibition appears to be mediated to a great part via the immunoglobulin Fab-portion. However, the exact mechanisms and potential microglial recognition molecules involved remain elusive. A broad range of specialized receptors have been implied in the phagocytosis of apoptotic cells and the subsequent modulation of phagocyte immune functions (Fadok et al., 2001). Lectin-, integrin- and phosphatidylserine-dependent mechanisms have been described in the phagocytosis of apoptotic targets by rodent microglia in vitro (De Simone et al., 2002; Witting et al., 2000). IVIg were recently demonstrated to inhibit leukocyte adhesion by antibodies against the RGD adhesion motif (Vassilev et al., 1999). In our system, RGDS peptides did not specifically inhibit microglial phagocytosis of apoptotic thymocytes, arguing against an integrin-mediated recognition/uptake mechanism (data not shown). Also, complement-components have been demonstrated in the phagocytosis of apoptotic cells (Fadok et al., 2001). Since in our study sera were heat-inactivated and all phagocytosis experiments were performed under serumfree conditions, microglial phagocytosis was not dependent on complement factors. Whatever mechanism involved, the inhibition of microglial phagocytosis of apoptotic cells by IVIg appears to be very potent, since even the strong phagocytosis-promoting effect of IFN-g was largely reversed by IVIg. An interference with IFN-g stimulation by anti-interferon antibodies in IVIg (Ross et al., 1995) was excluded by sequential IFN-g and IVIg pretreatment (data not shown). The ‘‘baseline’’ phagocytosis of non-steroid-treated cells and its inhibition by IVIg can partly be explained by the minor proportion of apoptotic cells inevitably present in the cell preparations. IVIg-mediated inhibition of unspecific phagocytosis mechanisms could
additionally play a role (Stangel et al., 2000b). Recently, IVIg have been demonstrated to increase Fc-receptor-mediated PNS-myelin phagocytosis by macrophages, while microglial CNS-myelin phagocytosis was not affected (Kuhlmann et al., 2002). However, the in vivo significance of these findings is unknown (Stangel et al., 2000b). Our data indicate that in autoimmune CNS-inflammation IVIg could interfere with the removal of the inflammatory infiltrate, at least during stages with a high prevalence of apoptotic inflammatory cells. Whether IVIg also inhibit the phagocytosis of apoptotic neurons and oligodendrocytes is currently unknown. In addition to several other presumed mechanisms of action, GLAT has also been demonstrated to alter macrophage effector functions in vitro (Siglienti et al., 2000). Here, we could not demonstrate an effect of GLAT on microglial phagocytosis of apoptotic or non-corticosteroid-treated thymocytes. MP increases T-cell apoptosis in situ in EAE but does not appear to affect glial cells (Schmidt et al., 2000). Moreover, glucocorticosteroids promote the phagocytosis of apoptotic granulocytes by human monocyte-derived macrophages in vitro (Liu et al., 1999). Here, MP led to rapid microglial apoptosis in vitro. Previous studies have shown a reduction in the number of rodent corpus callosum microglia after glucocorticosteroid injections (Wu et al., 2001). Thus, the effects of MP-pulse therapy during EAE on potential microglial apoptosis and possible influences on the phagocytosis of apoptotic cells merit further investigations. In conclusion, our data add to the growing notion that, in addition to peripheral mechanisms at least under conditions of an impaired blood – brain barrier, IVIg could also exert local immunomodulatory effects in the inflamed CNS. However, the complex interplay between these mechanisms remains to be elucidated in vivo.
Acknowledgements The authors thank Annette Horn for excellent technical support. We are indebted to Dr. Jack Antel for many stimulating discussions. We thank Dr. Martin Stangel for his expert advice on IVIg action on glial cells and Prof. Ioannis Milonas for his continuous support of C.P. Supported by grants from the Deutsche Forschungsgemeinschaft (DFG Go 459/8-3), funds from the state of Bavaria and a fellowship grant of the European Neurological Society to C.P.
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A. Chan et al. / Journal of Neuroimmunology 135 (2003) 161–165 Phagocytic clearance of apoptotic inflammatory cells by human and rodent microglia and effects of different immunomodulatory agents. Glia S1, S82. De Simone, R., Ajmone-Cat, M.A., Nicolini, A., Minghetti, L., 2002. Expression of phosphatidylserine receptor and down-regulation of proinflammatory molecule production by its natural ligand in rat microglial cultures. J. Neuropathol. Exp. Neurol. 61, 237 – 244. Fadok, V.A., Bratton, D.L., Henson, P.M., 2001. Phagocyte receptors for apoptotic cells: recognition, uptake, and consequences. J. Clin. Invest. 108, 957 – 962. Kazatchkine, M.D., Kaveri, S.V., 2001. Immunomodulation of autoimmune and inflammatory diseases with intravenous immune globulin. N. Engl. J. Med. 345, 747 – 755. Kuhlmann, T., Wendling, U., Nolte, C., Zipp, F., Maruschak, B., Stadelmann, C., Siebert, H., Bru¨ck, W., 2002. Differential regulation of myelin phagocytosis by macrophages/microglia, involvement of target myelin, Fc receptors and activation by intravenous immunoglobulins. J. Neurosci. Res. 67, 185 – 190. Liu, Y., Cousin, J.M., Hughes, J., van Damme, J., Seckl, J.R., Haslett, C., Dransfield, I., Savill, J., Rossi, A.G., 1999. Glucocorticoids promote nonphlogistic phagocytosis of apoptotic leukocytes. J. Immunol. 162, 3639 – 3646. Magnus, T., Chan, A., Savill, J., Toyka, K.V., Gold, R., 2002. Phagocytosis of apoptotic, inflammatory lymphocytes in the central nervous system (CNS) by microglia and its functional implications. J. Neuroimmunol. 130, 1 – 9. Nguyen, K.B., Pender, M.P., 1998. Phagocytosis of apoptotic lymphocytes by oligodendrocytes in experimental autoimmune encephalomyelitis. Acta Neuropathol. 95, 40 – 46. Pender, M.P., Rist, M.J., 2001. Apoptosis of inflammatory cells in immune control of the nervous system: role of glia. Glia 36, 137 – 144. Ross, C., Svenson, M., Hansen, M.B., Vejlsgaard, G.L., Bendtzen, K., 1995. High avidity IFN-neutralizing antibodies in pharmaceutically prepared human IgG. J. Clin. Invest. 95, 1974 – 1978. Schmidt, J., Gold, R., Zettl, U.K., Hartung, H.P., Toyka, K.V., 2000. T-cell apoptosis in situ in experimental autoimmune encephalomyelitis following methylprednisolone pulse therapy. Brain 123, 1431 – 1441.
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Siglienti, I., Toyka, K.V., Scarpini, E., Jung, S., 2000. Copolymer-1 effects on cytokine production by Lewis rat peritoneal macrophages. J. Neurol. 247, III/203. Stangel, M., Compston, A., 2001. Polyclonal immunoglobulins (IVIg) modulate nitric oxide production and microglial functions in vitro via Fc receptors. J. Neuroimmunol. 112, 63 – 71. Stangel, M., Compston, A., Scolding, N.J., 2000a. Oligodendroglia are protected from antibody-mediated complement injury by normal immunoglobulins (‘‘IVIg’’). J. Neuroimmunol. 103, 195 – 201. Stangel, M., Joly, E., Scolding, N.J., Compston, D.A., 2000b. Normal polyclonal immunoglobulins (‘IVIg’) inhibit microglial phagocytosis in vitro. J. Neuroimmunol. 106, 137 – 144. Vassilev, T., Kazatchkine, M.D., Van Huyen, J.P.D., Mekrache, M., Bonnin, E., Mani, J.C., Lecroubier, C., Korinth, D., Baruch, D., Schriever, F., Kaveri, S., 1999. Inhibition of cell adhesion by antibodies to ArgGly-Asp (RGD) in normal immunoglobulin for therapeutic use (intravenous immunoglobulin IVIg). Blood 93, 3624 – 3631. Warrington, A.E., Asakura, K., Bieber, A., Ciric, B., van Keulen, V., Kaveri, S.V., Kyle, R.A., Pease, L.R., Rodriguez, M., 2000. Human monoclonal antibodies reactive to oligodendrocytes promote remyelination in a model of multiple sclerosis. Proc. Natl. Acad. Sci. 97, 6820 – 6825. Wiles, C.M., Brown, P., Chapel, H., Guerrini, R., Hughes, R.A.C., Martin, T.D., McCrone, P., Newsom-Davis, J., Palace, J., Rees, J.H., Rose, M.R., Scolding, N., Webster, A.D.B., 2002. Intravenous immunoglobulin in neurological disease: a specialist review. J. Neurol. Neurosurg. Psychiatry 72, 440 – 448. 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. Wu, C.H., Chien, H.F., Chang, C.Y., Chen, S.H., Huang, Y.S., 2001. Response of amoeboid and differentiating ramified microglia to glucocorticoids in postnatal rats: a lectin histochemical and ultrastructural study. Neurosci. Res. 40, 235 – 244.
Short communication
Effects of polyclonal immunoglobulins and other immunomodulatory agents on microglial phagocytosis of apoptotic inflammatory T-cells Andrew Chan *,1, Christina Papadimitriou 1,2, Wolfgang Graf, Klaus V. Toyka, Ralf Gold Department of Neurology, Clinical Research Group for Multiple Sclerosis and Neuroimmunology, Julius-Maximilians-University, D-97080 Wu¨rzburg, Germany Received 3 September 2002; received in revised form 25 November 2002; accepted 25 November 2002
Abstract T-cell apoptosis in the CNS is an effective mechanism for the noninflammatory resolution of autoimmune T-cell infiltrates. Ingestion of apoptotic leukocytes by microglia results in an efficient clearance of the inflammatory infiltrate, followed by a profound downregulation of proinflammatory phagocyte immune functions. The effects of different immunomodulatory agents on Lewis rat microglial phagocytosis of apoptotic autologous thymocytes or myelin-basic protein (MBP)-specific, encephalitogenic T-cells were investigated using a standardized, light microscopical in vitro phagocytosis assay. Pretreatment of microglia with polyclonal 7S immunoglobulins (IVIg) decreased the phagocytosis of apoptotic thymocytes by 38.2% ( p < 0.0001). Also, immunoglobulin F(abV)2 fragments decreased microglial phagocytosis, suggesting an Fc receptor-independent mechanism. Similar results were obtained using MBP-specific T-cells. Pretreatment of microglia with IFN-g increased the phagocytosis of apoptotic cells by 65.4%, which was to a large extent counteracted by IVIg. Glatiramer acetate (GLAT) did not exert an effect on microglial phagocytosis, while methylprednisolone (MP) induced microglial apoptosis in vitro. These results indicate that IVIg has a high potential to inhibit microglial phagocytosis of apoptotic inflammatory T-cells even under proinflammatory conditions and extend our view of the complex immunomodulatory effects of IVIg. D 2002 Elsevier Science B.V. All rights reserved. Keywords: T-cell apoptosis; Multiple sclerosis; Experimental autoimmune encephalomyelitis; Glatiramer acetate; Glucocorticosteroids
1. Introduction Apoptosis of 30 –50% of all invading T-cells represents a crucial mechanism in the termination of autoimmune T-cellmediated inflammation in the human and rodent CNS, contributing to clinical recovery in experimental autoimmune encephalomyelitis (EAE) and acute disseminated leukoencephalomyelitis in man (ADEM) (Bauer et al., 2001; Pender and Rist, 2001). A key event in the resolution of an inflammatory infiltrate is the nonphlogistic and thus safe phagocytic clearance of
* Corresponding author. Neurologische Universita¨tsklinik, JosefSchneider-Straße 11, D-97080 Wu¨rzburg, Germany. Tel.: +49-931-20124621; fax: +49-931-201-23488. E-mail address: [email protected] (A. Chan). 1 Equally contributing authors. 2 Present address: Department of Neurology, University Clinic, Ahepa Hospital, Thessaloniki, Greece.
apoptotic leukocytes by tissue-specific phagocytes (Fadok et al., 2001). Phagocytosis of apoptotic lymphocytes by macrophages/microglia, oligodendrocytes and astrocytes has been described in situ in Lewis rat EAE (Nguyen and Pender, 1998). Lewis rat microglia efficiently phagocytoses apoptotic, encephalitogenic MBP-specific T-cells in vitro, differentially regulated by Th1-/Th2-type cytokines (Chan et al., 2001). The phagocytosis of apoptotic T-cells by Lewis rat microglia is more efficient than by astrocytes and leads to a profound downregulation of microglial immune functions, making this process an attractive target for therapeutic interventions (Magnus et al., 2002). The immunomodulatory agent interferon-beta augments phagocytosis specifically of apoptotic inflammatory cells, and also adult human microglia obtained from normal brain tissue phagocytoses apoptotic inflammatory cells in vitro (Chan et al., 2002). Here we set out to investigate the effects of other therapeutically used immunomodulatory agents on Lewis rat microglial phagocytosis of apoptotic inflammatory cells.
0165-5728/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0165-5728(02)00433-2
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A. Chan et al. / Journal of Neuroimmunology 135 (2003) 161–165
2. Materials and methods 2.1. Cell culture All cell culture media and supplements were obtained from Gibco/BRL (Eggenstein, Germany) unless otherwise noted. Microglial cells from neonatal Lewis rats (P0 –P2, Charles River, Sulzfeld, Germany) with a purity of consistently >97% were isolated as described before (Chan et al., 2001). Apoptosis of autologous thymocytes and of the MBPspecific, encephalitogenic CD4+ T-cell line MBP13 was induced by methylprednisolone (MP, Aventis Pharma, Bad Soden, Germany) as described in detail before (Chan et al., 2001). MP-treated thymocytes had a proportion of 37.8 F 2.6% (mean F S.E.M.) annexin V single-positive (early apoptotic) and 5.6 F 0.6% annexin V/PI-positive (late apoptotic/necrotic) cells, untreated control thymocytes were 12 F 2.6% annexin V-positive and 3.2 F 0.5% annexin V/PIpositive (Chan et al., 2001). The proportion of viable thymocytes or MBP13 cells excluding trypan blue was consistently >97%. 2.2. In vitro phagocytosis assay The standardized, microscopically quantified microglial phagocytosis assay has been described and illustrated in detail before (Chan et al., 2001). Four hundred microliters of a 0.75 106/ml suspension of microglial cells per well was seeded in 48-well plates (Costar) and cultured overnight at 37 jC/5% CO2 (BME, 10% FCS (Sigma Aldrich Chemie, Steinheim, Germany), 50 U/ml penicillin, 50 Ag/ ml streptomycin). Triplicate wells of microglial cells were then incubated with the respective concentrations of IVIg (SandoglobulinR, Novartis Pharma, Nu¨rnberg, Germany), F(abV)2 fragments (GammaveninR, Aventis Behring, Marburg, Germany), glatiramer acetate (GLAT, TEVA Pharma/ Aventis Pharma), MP or BME for 20– 24 h. Immunoglobulin preparations were dialyzed (H2O or BME) to remove stabilising agents that are potentially toxic in cell culture. Human albumin (Octapharma, Langenfeld, Germany; DRK Blutspendedienst, Baden-Baden, Germany) or ovalbumin (Sigma) were used as control proteins, respectively. In case of combined IFN-g (30 U/ml, R&D, Minneapolis, MN, USA))/IVIg pretreatment, microglia was either preincubated simultaneously or in another set of experiments sequentially treated with IFN-g (8 h) followed by IVIg (12 h). For RGDS/RGES peptide inhibition experiments, target cells were preincubated with the peptides (1– 2 mM, BME, 15 min, 4 jC) and subsequently added to the microglia without further washing. Thymocytes (500 Al, 20 106/ml in BME) or MBP13 T-cells (500 Al, 10 106/ ml) were co-cultured with the microglia (2 h, 37 jC, 5% CO2) followed by vigorous washing with cold PBS (4 jC) (Chan et al., 2001). After trypsinization, a separate cytocentrifuge preparation was obtained for each well and stained with May-Giemsa (Merck, Darmstadt, Germany)
(Chan et al., 2001). An average of 500 microglial cells per slide were counted in a blinded fashion by light microscopy. In some experiments, data is additionally given as phagocytic index (percent of phagocytosing microglia multiplied with the average number of ingested target cells per microglia) (Chan et al., 2001). All values are expressed as mean F S.E.M. Statistical significance was evaluated using Student’s t-test (GraphPad Software, San Diego, USA).
3. Results 3.1. IVIg and F(abV)2 fragments decrease microglial phagocytosis of apoptotic inflammatory cells As reported before, microglia has a high capacity to phagocytose apoptotic thymocytes or CNS autoantigenspecific, encephalitogenic T-cells in contrast to non-apoptotic target cells (Chan et al., 2001) (Figs. 1A and 2). As illustrated in Figs. 1A,B and 2, IVIg pretreatment (20 mg/ ml) decreased the phagocytosis of apoptotic thymocytes by 38.2 F 5.2% (mean F S.E.M.) in comparison to untreated microglia ( p < 0.0001). Pretreatment with human albumin (HA) did not exert an effect (Fig. 2). The decrease of phagocytosis was dose-dependent and reached a plateau with 20 mg/ml IVIg (percent inhibition, 10 mg/ ml: 26.3 F 6.25, p < 0.01; 30 mg/ml: 39.5 F 7.2%, p < 0.001). IVIg not only decreased the phagocytosis of corticosteroid-treated, apoptotic target cells, but also the much lower baseline phagocytosis of nontreated thymocytes, albeit to a lesser extent (Fig. 2, 27.5 F 3.4%, p < 0.0001). The stronger inhibitory IVIg effect on phagocytosis of apoptotic thymocytes was even more pronounced in the phagocytic index, which reflects the phagocytic capacity of individual microglial cells (IVIg 20 mg/ml, percent inhibition for apoptotic thymocytes: 32.6 F 7.5%, p < 0.001; for non-corticosteroid-treated cells: 18.2 F 6%, p < 0.05). IVIg (20 mg/ml) also decreased the phagocytosis of corticosteroid-treated, apoptotic, encephalitogenic MBP-specific MBP13 T-cells (percent inhibition 20.7 F 14.7%) and of non-corticosteroid-treated T-cells (13.6 F 5.6%). To investigate which portion of IVIg mediated the inhibition of phagocytosis, microglia was pretreated using F(abV)2 immunoglobulin fragments. Again, F(abV)2-mediated inhibition was more pronounced for the uptake of apoptotic thymocytes (49.1 F 3%, p < 0.0001) than for non-corticosteroid-treated cells (17.8 F 1.6%, p < 0.001). F(abV)2 fragments also decreased microglial phagocytosis of apoptotic MBP13 T-cells by 48.3 F 7% ( p < 0.0001), whereas no clear inhibition could be observed for non-glucocorticosteroidtreated T-cells. These results indicated that the suppression of phagocytosis of apoptotic and non-apoptotic cells was at least to a great part independent of Fc-receptor-mediated mechanisms.
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Fig. 2. Phagocytosis of autologous thymocytes by untreated and IVIgpretreated microglia. Microglia had a higher capacity for the uptake of corticosteroid-treated, apoptotic (+) thymocytes than for non-corticosteroidtreated ( ) thymocytes. IVIg decreased phagocytosis of apoptotic target cells and to a lesser extent of non-corticosteroid-treated cells in comparison to the untreated microglia, while human albumin (HA) did not have an effect on phagocytosis. Phagocytosis rate: given as percentage of the mean of the untreated controls (+) + S.E.M. 42.1 F 4.7% (mean F S.E.M.) of the untreated microglia were capable of phagocytosing apoptotic thymocytes. Five independent experiments, each performed in triplicates.
the phagocytic index by 88.4 F 29% ( p < 0.05), this effect was reduced by IVIg (20 mg/ml) by 69.4 F 2% ( p < 0.05). 3.3. Glatiramer acetate (GLAT) does not have an effect on microglial phagocytosis while methylprednisolone (MP) induces microglial apoptosis in vitro Pretreatment with GLAT (10 – 50 Ag/ml) did not show any specific effects on microglial phagocytosis of apoptotic or Fig. 1. Photomicrographs of untreated (A) or IVIg-pretreated (20 mg/ml) Lewis rat microglia (B) after 2 h interaction with autologous apoptotic thymocytes, which show typical apoptotic morphology with condensed chromatin (arrows). May-Giemsa stain. Bar = 10 Am.
3.2. IVIg partially counteracts the phagocytosis-promoting effect of interferon-gamma (IFN-c) As reported before, IFN-g increases microglial phagocytosis of apoptotic thymocytes, whereas the uptake of nonapoptotic target cells is not altered (Chan et al., 2001). IFN-g (30 U/ml) augmented the microglial phagocytosis rate for apoptotic thymocytes by 65.4 F 18.5% above the untreated controls (Fig. 3, p < 0.01), similar to values reported previously (Chan et al., 2001). Simultaneous preincubation of microglia with IFN-g and IVIg partially reversed this effect in an IVIg dose-dependent manner (percent inhibition of the IFN-g-augmented phagocytosis rate, IVIg 10 mg/ml: 33.2 F 8.4%; IVIg 20 mg/ml: 49.4 F 2.8%, p < 0.05). No change was observed with the combination of IFN-g and HA (Fig. 3). The inhibitory effect was even more pronounced using the phagocytic index. Whereas IFN-g alone increased
Fig. 3. Microglial phagocytosis of apoptotic thymocytes after pretreatment with IFN-g, IFN-g/IVIg or IFN-g/human albumin. IFN-g (30 IU/ml) increased the phagocytosis rate for apoptotic target cells (+), which was partially counteracted by the combination with IVIg (IFN-g/IVIg, 20 mg/ml). Combined pretreatment with IFN-g and human albumin (IFN-g/HA, 20 mg/ ml) did not alter the phagocytosis-promoting effect of IFN-g. Phagocytosis rate: given as percentage of the mean of the untreated controls (+) + S.E.M. 34.9 F 5.9% (mean F S.E.M.) of the untreated microglia were capable of phagocytosing apoptotic thymocytes. Two independent experiments, each performed in triplicates.
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non-corticosteroid-treated thymocytes in comparison to ovalbumine (data not shown). Pretreatment of microglia with MP (50 –1000 nM) resulted in decreased phagocytosis of thymocytes. Immunohistochemistry demonstrated a profound induction of microglial apoptosis even by low concentrations of MP with all cells showing annexin V/PI positivity after 3 h of MP treatment (200 nM, data not shown).
4. Discussion Although high dose IVIg are widely used in neurological diseases of presumed autoimmune etiology, their pleiotropic mechanisms of action are only incompletely understood (Kazatchkine and Kaveri, 2001; Wiles et al., 2002). More recently, a possible influence of IVIg also on local CNS immune reactions and remyelination has been suggested (Stangel et al., 2000a; Stangel and Compston, 2001, Warrington et al., 2000). Here, we have demonstrated that IVIg in concentrations corresponding to serum levels during IVIg treatment inhibit microglial phagocytosis of apoptotic thymocytes as well as MBP-specific, encephalitogenic T-cells. This inhibition appears to be mediated to a great part via the immunoglobulin Fab-portion. However, the exact mechanisms and potential microglial recognition molecules involved remain elusive. A broad range of specialized receptors have been implied in the phagocytosis of apoptotic cells and the subsequent modulation of phagocyte immune functions (Fadok et al., 2001). Lectin-, integrin- and phosphatidylserine-dependent mechanisms have been described in the phagocytosis of apoptotic targets by rodent microglia in vitro (De Simone et al., 2002; Witting et al., 2000). IVIg were recently demonstrated to inhibit leukocyte adhesion by antibodies against the RGD adhesion motif (Vassilev et al., 1999). In our system, RGDS peptides did not specifically inhibit microglial phagocytosis of apoptotic thymocytes, arguing against an integrin-mediated recognition/uptake mechanism (data not shown). Also, complement-components have been demonstrated in the phagocytosis of apoptotic cells (Fadok et al., 2001). Since in our study sera were heat-inactivated and all phagocytosis experiments were performed under serumfree conditions, microglial phagocytosis was not dependent on complement factors. Whatever mechanism involved, the inhibition of microglial phagocytosis of apoptotic cells by IVIg appears to be very potent, since even the strong phagocytosis-promoting effect of IFN-g was largely reversed by IVIg. An interference with IFN-g stimulation by anti-interferon antibodies in IVIg (Ross et al., 1995) was excluded by sequential IFN-g and IVIg pretreatment (data not shown). The ‘‘baseline’’ phagocytosis of non-steroid-treated cells and its inhibition by IVIg can partly be explained by the minor proportion of apoptotic cells inevitably present in the cell preparations. IVIg-mediated inhibition of unspecific phagocytosis mechanisms could
additionally play a role (Stangel et al., 2000b). Recently, IVIg have been demonstrated to increase Fc-receptor-mediated PNS-myelin phagocytosis by macrophages, while microglial CNS-myelin phagocytosis was not affected (Kuhlmann et al., 2002). However, the in vivo significance of these findings is unknown (Stangel et al., 2000b). Our data indicate that in autoimmune CNS-inflammation IVIg could interfere with the removal of the inflammatory infiltrate, at least during stages with a high prevalence of apoptotic inflammatory cells. Whether IVIg also inhibit the phagocytosis of apoptotic neurons and oligodendrocytes is currently unknown. In addition to several other presumed mechanisms of action, GLAT has also been demonstrated to alter macrophage effector functions in vitro (Siglienti et al., 2000). Here, we could not demonstrate an effect of GLAT on microglial phagocytosis of apoptotic or non-corticosteroid-treated thymocytes. MP increases T-cell apoptosis in situ in EAE but does not appear to affect glial cells (Schmidt et al., 2000). Moreover, glucocorticosteroids promote the phagocytosis of apoptotic granulocytes by human monocyte-derived macrophages in vitro (Liu et al., 1999). Here, MP led to rapid microglial apoptosis in vitro. Previous studies have shown a reduction in the number of rodent corpus callosum microglia after glucocorticosteroid injections (Wu et al., 2001). Thus, the effects of MP-pulse therapy during EAE on potential microglial apoptosis and possible influences on the phagocytosis of apoptotic cells merit further investigations. In conclusion, our data add to the growing notion that, in addition to peripheral mechanisms at least under conditions of an impaired blood – brain barrier, IVIg could also exert local immunomodulatory effects in the inflamed CNS. However, the complex interplay between these mechanisms remains to be elucidated in vivo.
Acknowledgements The authors thank Annette Horn for excellent technical support. We are indebted to Dr. Jack Antel for many stimulating discussions. We thank Dr. Martin Stangel for his expert advice on IVIg action on glial cells and Prof. Ioannis Milonas for his continuous support of C.P. Supported by grants from the Deutsche Forschungsgemeinschaft (DFG Go 459/8-3), funds from the state of Bavaria and a fellowship grant of the European Neurological Society to C.P.
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