Graefes Arch Clin Exp Ophthalmol (2010) 248:985990 DOI 10.

1007/s00417-009-1289-x

BASIC SCIENCE

IL-2 and IFN-gamma in the retina of diabetic rats

Siv Johnsen-Soriano & Mara Sancho-Tello & Emma Arnal & Amparo Navea & Enrique Cervera & Francisco Bosch-Morell & Maria Miranda & Francisco Javier Romero

Received: 15 May 2009 / Revised: 30 November 2009 / Accepted: 20 December 2009 / Published online: 6 March 2010 # Springer-Verlag 2010

Abstract Background The pathophysiology of the early events leading to diabetic retinopathy is not fully understood. It has been suggested that Inflammatory processes are involved in the development of the disease; however, the concentrations of tissue retinal inflammatory mediators and their possible alteration in diabetic retinopathy have not been described. The aim of this work was to study T-helper cell cytokine and chemokine profiles, and tyrosine nitration in retinal tissue of diabetic rats.

A financial relationship with the Conselleria de Educacin exists in form of a Grant. The authors have full control of all primary data, and we agree to allow Graefes Archive for Clinical and Experimental Ophthalmology to review our data upon request. S. Johnsen-Soriano : M. Sancho-Tello : E. Arnal : A. Navea : F. Bosch-Morell : F. Javier Romero (*) Fundacin Oftalmolgica del Mediterrneo (FOM), Bifurcacin Pio Baroja-General Avils, s/n 46015 Valencia, Spain e-mail: jromero@uch.ceu.es S. Johnsen-Soriano : F. Bosch-Morell : M. Miranda : F. Javier Romero Departamento de Fisiologa, Farmacologa y Toxicologa, Universidad CEU-Cardenal Herrera, Avda Seminario s/n, Moncada, 46113 Valencia, Spain M. Sancho-Tello Departamento de Patologa, Facultad de Medicina y Odontologa, Universidad de Valencia, Avda. Blasco Ibaez 15, 46010 Valencia, Spain E. Cervera Servicio de Oftalmologa, Hospital General Universitario, 46015 Valencia, Spain

Methods Cytokines (interleukin IL-1a, IL-1b, IL-2, IL-4, IL-6, IL-10, TNFa, GM-CSF, IFN-g), chemokines (MIP-1a, MIP-2, MIP-3a, MCP-1, GRO/KC, RANTES, Fractalkine), and tyrosine nitration were measured in retinal homogenate obtained from LongEvans rats after 5 months of experimental diabetes. Results The T-helper type 1 cytokines IL-2 and INFgamma, in addition to NO production (measured as nitrotyrosine), were found to be significantly elevated in diabetic rat retina homogenates. None of the other cytokines and chemokines studied were affected by the diabetic condition. Conclusions Immunoregulatory cytokines belonging to the Th-1 group (IL-2 and IFN-gamma) were increased in the retina of experimental diabetic rats. Moreover, the nitrotyrosine formation (as an expression of increased NO production) was significantly elevated in the diabetic retina, supporting the concept of an inflammatory element in the development of diabetic retinopathy. Keywords Diabetic retinopathy . IL-2 . IFN-gamma . Chemokine . Cytokine

Introduction Diabetic retinopathy is a progressive disease induced by chronic exposure to high blood glucose levels, e.g. hyperglycemia, and it is generally recognized as a vascular disease like many other diabetes-related alterations. Extensive research has been carried out in order to develop therapies for diabetic retinopathy; however, the pathogenesis of diabetic retinopathy has not been clearly elucidated due to the complicated and intricate biochemical and pathophysiological aspects of the disease. In addition to metabolic control, current therapy for diabetic retinopathy

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Graefes Arch Clin Exp Ophthalmol (2010) 248:985990

includes laser photocoagulation and surgery, while future therapies are based on the pathophysiology of the disease, such as anti-VEGF, anti-angiogenesis, and antiinflammatory pathways [1]. Animal models, as well as clinical studies, have reported that inflammation contributes to the development of diabetic retinopathy [2]. Furthermore, increased leucostasis has been reported in retinas of diabetic animals [3], as well as increased expressions of ICAM-1 [4] and platelet activation [5]. Elevated levels of TNF alpha in epiretinal membranes have been reported in diabetic patients [6], together with increased levels of the proinflammatory cytokine IL-1b in retinal cell cultures exposed to high glucose concentrations [7, 8]. Furthermore, various clinical and experimental studies have reported elevated concentrations of cytokines such as IL8, IL-2, IL-1a IL-6, INF-gamma in samples from serum, vitreous, aqueous humor, and epiretinal membranes from subjects with diabetic retinopathy [915]. However, diabetes mellitus is a disease that affects many tissues and organs, causing retinopathy, nephropathy, neuropathy, cardiovascular diseases, peripheral vascular diseases, and stroke. The analysis of cytokine serum markers may be too. To our knowledge, the T-helper cell cytokine profile in the retina of diabetic rats has not yet been previously reported. In addition to cytokines, nitrotyrosine has been identified as an indicator of cell damage and inflammation, as a consequence of excessive production of nitric oxide (NO), a key inducer and regulator of the immune system activation [16]. In this study, we investigated tyrosine nitration (as an expression of NO production), and cytokine and chemokine concentrations in the retina of long-term diabetic rats. Appropriate levels of NO production are important in protecting an organ from nitrosative damage. NO is an intercellular messenger that performs a number of functions, including neurotransmission, vasodilation, inhibition of platelet aggregation, and modulation of leukocyte adhesion. NO has recently been shown to act as a potent cytotoxic effector molecule, as well as to play an important role in the pathogenesis of organ-specific autoimmunity. NO may also modulate the immune response by interfering with Th1/Th2 balance in autoimmune diseases [17]. It is essential to know the pathophysiology of the diabetic retinopathy and the factors at play during the development at the disease, in order to design new therapies. Based on earlier findings supporting the role of inflammatory elements in the development of diabetic retinopathy, the purpose of this study is to assess the proper retinal concentration and the possible alteration of several inflammatory mediators such as cytokines, chemokines, and NO in an experimental model of diabetic retinopathy.

Materials and methods Twelve male LongEvans rats (200220 g) were individually housed in a temperature- and humidity-controlled room with a 12-hour light/dark cycle, and were provided with food and water ad libitum. All animal manipulations were done according to international regulations of the European Economic Community (directive 86/608/CEE) and ARVO (Association for Research in Vision and Ophthalmology). Rats were randomly assigned to normal or diabetic group. Diabetes was induced with streptozotocin injection (55 mg/kg body weight, intraperitoneally), and only the rats with blood sugar levels higher than 20 mM 3 days after administration of streptozotocin were assigned to the diabetic group and included in the experiment. Rats were weighed 3 times a week. Diabetic rats were administered small insulin (lantus) doses (4 UI/kg) 3 times a week, simply to allow a slow weight gain while maintaining hyperglycemia (blood glucose levels more than 20 mM). Blood glucose levels were weekly controlled with a glucometer (Precision PCx; Medisence, Cambridge, UK). Rats were kept diabetic for 5 months and then anesthetized with ketamine (100 mg/kg body weight) and azepromazine (2.5 mg/kg body weight) and decapitated; the eyes were enucleated immediately. Then, retinas were dissected under a microscope and homogenized in prechilled 0.2 M potassium phosphate buffer, pH 7.0. Samples were kept frozen (80C) until biochemical assays were performed. Enzyme-linked immunosorbent assays (ELISA) (sample number in each group was five and two replicates) were performed to quantify different chemokines and cytokines in retinal rat homogenates, using Searchlight Rat Chemokine Array 1 and Cytokine Array (Pierce Biotechnology, Inc., Woburn, MA, USA). All procedures were performed according to the manufacturers manual (http://www.piercenet.com). Nitrotyrosine in retinal rat homogenates was quantified with an ELISA kit (A.G. Scientific Inc, San Diego, CA, USA). All procedures were performed according to the manufacturers manual. The results are presented as mean values SEM. Statistical significances were assessed by one-way ANOVA followed by the Duncan test. The level of significance was set at p0.05.

Results Confirmation of experimental diabetes Before treatment, there was no significant difference between any group in weight or random glucose levels.

Graefes Arch Clin Exp Ophthalmol (2010) 248:985990 Table 1 General characteristics of the experimental groups Blood glucose (mM) Control Diabetic 4.50.2 29.90.8 *
a

987

Chemokine levels in diabetic retina

b

Body weight (g) 4487.6 30117.3*

* p0.05 vs control. Data are means SEM or range of means. a throughout study; b at the end of the study. N=6 animals per group.

Five months after diabetes induction by streptozotocin injection, random blood glucose concentration in the diabetic group was significantly higher than in the control group (29.90.8 and 4.50.2 mM, p0.05) as shown in Table 1. As expected, body weight in the diabetic animals was somewhat lower than in the control group (Table 1). Gross ocular findings At the beginning of the third month after diabetes induction, early cataract formation was evident and progressed for 2 more months. Therefore, after 5 months of diabetes, a white cataract was present in the eyes of all diabetic rats (Fig. 1a), but in none of the control group (Fig. 1b). Cytokine levels in retinal homogenates Nine cytokines were analysed in retinal homogenates of control and diabetic rats (IL-1a, IL-1b, IL-2, IL-4, IL-6, IL-10, TNFa, GM-CSF, IFN-g) in order to study the effect of diabetes on the retinal concentration of these cytokines (Table 2, Fig. 2). Although a slight increase of cytokine IL-1b concentration in the diabetic group was detected, no statistical differences were found between any of the cytokine concentrations in the diabetic and the control group. The cytokines IL-6 and GM-CSF were found in both groups to be below the detection limit, 7 pmol/ml and 1 pg/ml respectively. The relevant findings were the significant increase of IL-2 and IFN-g concentrations in the diabetic condition (*p0.05 vs control, Fig. 2), indicating a T-helper type 1 response.
Fig. 1 Cataract formation. a Diabetic rat after 5 months with diabetes. b Control rat without changes in the lens

Seven chemokines (MIP-1a, MIP-2, MIP-3a, MCP-1, GRO/KC, RANTES, Fractalkine) were analysed in retinal homogenate of diabetic and control rats. In the chemokine assay, all chemokines analyzed were above the detection limit. As shown in Table 3, a decrease of MIP-1a and MCP-1 in the diabetic retina was observed when compared to controls; however, these decreases were not statistically different, due to great individual differences in these chemokine concentrations. Nitrotyrosine levels in diabetic retina The nitrotyrosine content in diabetic and control rats were mean nitrotyrosine level of 5.8 nM/mg protein, while the retina was 2-fold higher (14.7 significantly different from the control) (Fig. 3). retinal homogenate from analyzed by ELISA. The the control retinas was mean content in diabetic nM/mg protein), and was control retinas (p0.05 vs

Discussion Herein, we report the cytokine and chemokine levels of retinal tissue in control and diabetic rats. Nine cytokines and seven chemokines were analysed in retinal homogenates of control and diabetic rats. Earlier studies investigating the cytokine expression in diabetic retinopathy have shown that TNF-a is increased in serum [18], vitreous [15] and epiretinal membranes [6] of patients with diabetic retinopathy, and in retinal endothelial cells [19]. We have found no increase of TNF-a content in the rat diabetic retina (Table 2), in agreement with former studies that had suggested a somehow tissue-specific lack of expression of TNF-a in the retina of diabetic subjects [18]. Although other studies have reported an increase in IL-1b in different retinal cell lines cultured in the presence of glucose [8, 19, 20], the only change observed in vivo in

988 Table 2 Chemokine array of retina homogenate Chemokines MIP-1a MIP-2 MIP-3a MCP-1 GRO/KC RANTES Fractalkine Control 0.660.27 0.880.50 2.320.80 27.449.20 2.511.25 2.30.78 18.472.3 Diabetes 0.380.23 0.850.27 2.700.50 12.516.20 1.980.99 3.350.75 19.723.25

Graefes Arch Clin Exp Ophthalmol (2010) 248:985990 Table 3 Cytokine array of retina homogenate Cytokines IL-1a IL-1b IL- 4 IL- 6 IL-10 TNFa GM-CSF Control 1.310.33 4.290.86 5.220.68 Non detectable 2.460.86 8.60.92 Non detectable Diabetes 1.660.17 6.740.76 6.021.30 Non detectable 1.850.55 7.750.60 Non detectable

All chemokines expressed as pg/ml. Data are means SEM.

All cytokines expressed as pg/ml. Data are means SEM.

diabetic rat retina was at the mRNA levels of IL-1b [18, 21], not disagreeing with our results (cf. Table 2). Herein, we show an increase in IL-2 and INF-g levels in diabetic retinal homogenates by using a protein array (Fig. 2). IL-2 is produced mainly by activated T helper (Th, CD4+) lymphocytes, and the major action of IL-2 is on T lymphocytes, NK cells, B lymphocytes, and monocytes [22]. IL-2 activates T-cell growth in vitro, while its role in vivo is controversial [23]. Interestingly, IL-2 inhibits growth of certain human tumour cells, while proliferation of others remains intact or is even stimulated [22]. IL-2 induces cell growth and differentiation with augmentation of cellular activities, and it may directly affect its target cells, or indirectly induce the expression of endogenous cytokines, such as INF-g [24]. Augmented concentrations of IL-2 and IFN-g in serum and other body fluids occur in diseases of neoplastic, infective, or autoimmune nature [25, 26], but to our knowledge it has not been investigated in relationship to diabetic retinopathy. Earlier studies have only reported increased levels of the soluble receptors for IL-2 in serum [27, 28] and in epiretinal membranes [10] from eyes of patients with proliferative diabetic retinopathy; however, neither IL-2 nor IFN-g increases in the retinal tissue during experimental diabetic retinopathy have been reported so far. Geiger et al. reported that transgenic mice
40 35 Cytokines (pg/ml) 30 25 20 15 10 5 0 IL-2 Control Diabetes

expressing IFN-g in the retina developed inflammation of the eye and photoreceptor loss [29]. Furthermore, IFN-g has not been detected in vitreous or in aqueous fluid of patients with proliferative diabetic retinopathy [9]. Taken together with our results, it seems likely that the retina has a selective response to the diabetic insult. Since both IL-2 and IFN-g are classically part of the Th1 cytokine response, the results herein convincingly allow the proposal that in this experimental model of diabetic retinopathy, a retinal Th1 cytokine response occurs. The results showed that there were no differences in chemokine levels between diabetic and control retinas (Table 3). Previous studies in human samples have reported increased levels of MCP-1 and RANTES in vitreous and serum samples [11, 13, 14]. Our results also show a slight increase in RANTES and MCP-1 in the rat diabetic retina, in agreement with these human findings; however, no statistical differences between the diabetic and the control group could be established (Table 3). One possible explanation for this apparent discrepancy with our data could be that these observed increases of MCP-1 and RANTES in diabetes could be tissue-specific. In fact, Maier et al. [13] found that MCP-1 was increased in the vitreous, while MIP-1b and RANTES were increased in the serum of diabetic patients. The analysis of the retinal homogenate in our study showed an increase of nitrotyrosine concentration (Fig. 3),
20 18 16 14 12 10 8 6 4 2 0 Control Nitrotyrasine nM/mg protein

* *
INFg

Diabetes

Fig. 2 Cytokine in diabetic retina. Data are means SEM. * p0.05 vs control

Fig. 3 Nitrotyrosine levels in diabetic retina. Data are means SEM. * p0.05 vs control

Graefes Arch Clin Exp Ophthalmol (2010) 248:985990

989 6. Joussen AM, Poulaki V, Mitsiades N, Kirchhof B, Koizumi K, Dohmen S, Adamis AP (2002) Nonsteroidal anti-inflammatory drugs prevent early diabetic retinopathy via TNF-alpha suppression. Faseb J 16:438440 7. Kowluru RA, Odenbach S (2004) Role of interleukin-1beta in the pathogenesis of diabetic retinopathy. Br J Ophthalmol 88:1343 1347 8. Vincent JA, Mohr S (2007) Inhibition of caspase-1/interleukin1beta signaling prevents degeneration of retinal capillaries in diabetes and galactosemia. Diabetes 56:224230 9. Abu el Asrar AM, Maimone D, Morse PH, Gregory S, Reder AT (1992) Cytokines in the vitreous of patients with proliferative diabetic retinopathy. Am J Ophthalmol 114:731736 10. Tang S, Scheiffarth OF, Thurau SR, Wildner G (1993) Cells of the immune system and their cytokines in epiretinal membranes and in the vitreous of patients with proliferative diabetic retinopathy. Ophthalmic Res 25:177185 11. Meleth AD, Agron E, Chan CC, Reed GF, Arora K, Byrnes G, Csaky KG, Ferris FL 3rd, Chew EY (2005) Serum inflammatory markers in diabetic retinopathy. Invest Ophthalmol Vis Sci 46:42954301 12. Patel JI, Tombran-Tink J, Hykin PG, Gregor ZJ, Cree IA (2006) Vitreous and aqueous concentrations of proangiogenic, antiangiogenic factors and other cytokines in diabetic retinopathy patients with macular edema: Implications for structural differences in macular profiles. Exp Eye Res 82:798806 13. Maier R, Weger M, Haller-Schober EM, El-Shabrawi Y, Wedrich A, Theisl A, Aigner R, Barth A, Haas A (2008) Multiplex bead analysis of vitreous and serum concentrations of inflammatory and proangiogenic factors in diabetic patients. Mol Vis 14:637643 14. Murugeswari P, Shukla D, Rajendran A, Kim R, Namperumalsamy P, Muthukkaruppan V (2008) Proinflammatory cytokines and angiogenic and anti-angiogenic factors in vitreous of patients with proliferative diabetic retinopathy and Eales disease. Retina 28:817 824 15. Vijay SK, Mishra M, Kumar H, Tripathi K (2009) Effect of pioglitazone and rosiglitazone on mediators of endothelial dysfunction, markers of angiogenesis and inflammatory cytokines in type-2 diabetes. Acta Diabetol 46:2733 16. ter Steege JC, Koster-Kamphuis L, van Straaten EA, Forget PP, Buurman WA (1998) Nitrotyrosine in plasma of celiac disease patients as detected by a new sandwich ELISA. Free Radic Biol Med 25:953963 17. Singh VK, Mehrotra S, Narayan P, Pandey CM, Agarwal SS (2000) Modulation of autoimmune diseases by nitric oxide. Immunol Res 22:119 18. Gustavsson C, Agardh CD, Hagert P, Agardh E (2008) Inflammatory markers in nondiabetic and diabetic rat retinas exposed to ischemia followed by reperfusion. Retina 28:645652 19. Nehme A, Edelman J (2008) Dexamethasone inhibits high glucose-, TNF-alpha-, and IL-1beta-induced secretion of inflammatory and angiogenic mediators from retinal microvascular pericytes. Invest Ophthalmol Vis Sci 49:20302038 20. Kowluru RA, Odenbach S (2004) Role of interleukin-1beta in the development of retinopathy in rats: effect of antioxidants. Invest Ophthalmol Vis Sci 45:41614166 21. Krady JK, Basu A, Allen CM, Xu Y, LaNoue KF, Gardner TW, Levison SW (2005) Minocycline reduces proinflammatory cytokine expression, microglial activation, and caspase-3 activation in a rodent model of diabetic retinopathy. Diabetes 54:15591565 22. Olejniczak K, Kasprzak A (2008) Biological properties of interleukin 2 and its role in pathogenesis of selected diseasesa review. Med Sci Monit 14:RA179RA189 23. Antony PA, Paulos CM, Ahmadzadeh M, Akpinarli A, Palmer DC, Sato N, Kaiser A, Hinrichs CS, Klebanoff CA, Tagaya Y, Restifo NP (2006) Interleukin-2-dependent mechanisms of tolerance and immunity in vivo. J Immunol 176:52555266

suggesting an increased NO production in the rat diabetic retina. Increased NO has earlier been correlated with diabetic retinopathy [30], and a recent study reported that high glucose levels cause increased cell damage and NO generation in RPE cells, which requires the activation of p38MAPK and ERK [31]. Interestingly, the cellular signal transduction pathway for IL-2 also involves the participation of the Ras/ERK/MAPK pathway [3234]. Therefore, a possible sequence for the observed augmentation of the cytokines (IL-2 and INF-g) and NO production in our study could be that increased IL-2 levels activates IFN-g production which in turn increases NO production, a sequence recently described by Shio et al. [35]. Diabetic retinopathy has earlier been linked to apoptosis [36], and more recently a strong correlation between apoptosis and immunosuppression has also been suggested [3739]. A recent report has studied the molecular mechanism controlling this apoptotic cell-mediated immunosuppression, and the results revealed a novel mechanism through which apoptotic cells induce immunosuppression by increasing NO production via IFN-g induction [40]. This could also be applied to the experimental model herein, in view of the consistency of all conditions mentioned, i.e. increased nitrotyrosine as a result of enhanced NO production, together with increased levels of IL-2 and IFN-g in the diabetic retina. In summary, immunoregulatory cytokines belonging to the Th-1 group (IL-2 and IFN-g) were increased in retinal homogenate from rats with experimental diabetes. These findings, together with the evidence of a significant elevation of NO production in the diabetic retina (nitrotyrosine increase), strongly support the concept of an inflammatory element in the development of diabetic retinopathy. In view of the complexity of the immune responses and other exogenous factors that modulate immunity, further experimental and clinical studies are indispensable.

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