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Dysregulation of IL-10 and IL-12p40 in secondary progressive multiple sclerosis
Journal of Neuroimmunology 146 (2004) 209 – 215 www.elsevier.com/locate/jneuroim
Short communication
Dysregulation of IL-10 and IL-12p40 in secondary progressive multiple sclerosis Samantha S. Soldan, Ana Isabel Alvarez Retuerto, Nancy L. Sicotte, Rhonda R. Voskuhl * Department of Neurology, Reed Neurological Research Center, University of California School of Medicine, 750 Westwood Plaza, Los Angeles, CA 90095, USA Received 28 July 2003; received in revised form 3 October 2003; accepted 3 October 2003
Abstract Multiple sclerosis (MS) is a putative T helper 1 (Th-1) mediated inflammatory disorder of the central nervous system, and levels of proinflammatory and anti-inflammatory cytokines have been found to correlate with changes in MS relapses. However, it is unclear if cytokine profiles differ between relapsing – remitting (RRMS) versus secondary progressive (SPMS) disease stages. Cytokine production (IL-2, IL-4, IL-5, IL-10, IL-12p40, TNF-a, and IFN-g) was assessed by cytometric bead array (CBA) and intracellular cytokine staining from aCD3 antibody and mitogen stimulated peripheral blood mononuclear cells (PBMCs) from female RRMS and SPMS patients, and healthy controls. Significantly increased production of IL-12p40 and decreased production of IL-10 were observed in SPMS patients. Differences in immune responsiveness in RRMS and SPMS are important in the understanding of the evolution of the immunopathogenesis of the disease and for the development of disease type specific treatments. D 2003 Elsevier B.V. All rights reserved. Keywords: Multiple sclerosis; Cytokines; Th1/Th2
1. Introduction Multiple sclerosis is the most prevalent demyelinating disease of young adults with a clear gender bias involving an increased incidence in females (Duquette et al., 1992; Duquette and Girard, 1993; Whitacre et al., 1999). The most common form of MS is the relapsing – remitting MS (RRMS) phenotype that is characterized by disease exacerbations where new symptoms appear or existing symptoms become more severe. Most individuals who are initially diagnosed with RRMS will develop secondary progressive MS (SPMS) marked by the gradual accumulation of progressive disability. It is widely believed that T helper 1 (Th-1) mediated responses are involved in the pathogenesis of multiple sclerosis (MS) (Lu et al., 1993; Rieckmann et al., 1995; Beck et al., 1988). This is based on the association of MS with genes involved with the immune system, the immunopathology of the disease, the clinical response of MS
* Corresponding author. Tel.: +1-310-206-4636; fax: +1-310-2069801. E-mail address: [email protected] (R.R. Voskuhl). 0165-5728/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2003.10.033
patients to immunomodulatory and immunosuppressive treatments, and similarities with experimental immune-mediated demyelinating diseases in animals. According to the Th-1/Th-2 paradigm, Th-1 lymphocytes secrete proinflammatory cytokines (e.g. IFNg, TNFa), while Th-2 lymphocytes produce anti-inflammatory cytokines (e.g. IL-4, IL-5, IL-10) that promote humoral responses and confer protection from Th-1 mediated autoimmunity. While the counterregulatory roles of Th-1 and Th-2 cytokines are wellestablished in murine systems, this dichotomy is more ambiguous in humans (Sinigaglia et al., 1999). Nevertheless, the Th-1/Th-2 paradigm has been useful in understanding the mechanism of immunomodulatory therapies for MS (Duda et al., 2000; Rudick et al., 1998). As compared to RRMS, SPMS is characterized by fewer enhancing lesions on brain MRI and greater axonal degeneration (Trapp et al., 1998; Bjartmar et al., 2003; Trapp et al., 1999). Unfortunately, when patients transition from RRMS to SPMS, their disease often becomes refractory to immunomodulatory therapies. The disparate therapeutic effects of immunomodulatory therapy in these disease phases may reflect differences in the relative contribution of axonal pathology to disease course and/or differences in the immunopathogenesis of these disease phases. Some
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differences in immune dysregulation between RRMS and SPMS have been described (Balashov et al., 1997, 2000; Pelfrey et al., 2002; Sorensen and Sellebjerg, 2001; van Boxel-Dezaire et al., 1999). While recent studies have reported distinctly altered cytokine profiles in RRMS and SPMS, there is no consensus between these studies regarding the relationship between cytokine profiles and disease phenotype (Balashov et al., 2000; van Boxel-Dezaire et al., 1999; Pelfrey et al., 2002). In this study, cytokine production was determined in response to ex vivo stimulation. We focused exclusively on female subjects to assess differences in cytokine profiles between disease phases while eliminat-
ing the confounding effects of gender (Kim and Voskuhl, 1999; Nguyen et al., 2003; Pelfrey et al., 2002).
2. Materials and methods 2.1. Patient and control samples Peripheral blood mononuclear cells (PBMCs) were obtained from 12 female patients with clinically definite MS (6 RR and 6 SP) and 10 female, age-matched healthy controls (ND). MS patients and ND were screened by history
Fig. 1. Decreased IL-10 and increased IL-12p40 secretion in SPMS. Secreted cytokine levels (IL-2, IL-4, IL-5, IL-10, IL-12 p40, TNF-a, and IFN-g) were measured in tissue culture supernatant 48 h after stimulation with (a) soluble aCD3 and (b) PHA. (a) Decreased IL-10 production was observed in aCD3 stimulated SPMS PBMC compared to both RRMS ( p = 0.003) and ND ( p = 0.002). In addition, IL-12p40 production was increased SPMS compared to RRMS ( p = 0.003) and ND ( p = 0.003). (b) IL-10 production was also significantly decreased in SPMS PBMC stimulated with PHA. This decrease in IL-10 was observed in comparison to PBMC from RRMS ( p < 0.01) and ND ( p < 0.01). IL-12 was significantly increased in SPMS PBMC stimulated with PHA as compared to RRMS ( p = 0.028) and ND ( p = 0.04). The range of detection was 20 – 5000 pg/ml for each cytokine measured by CBA and 20 – 2000 for IL12p40. Bars represent intragroup mean. Asterisks: p < 0.05.
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and serologic studies and did not present evidence of chronic infectious, neoplastic, or other autoimmune diseases. MS patients had not received steroid treatment for at least 3 months or IFN-h or glatiramer acetate for 6 months prior. The mean age of the MS patients was 44 years (range, 28 –50 years) while the mean age of the ND group was 38 years (range, 21 –58). The mean Expanded Disability Status Scale (EDSS) score was 3.3 (range, 1.0 – 6.5) with clear differences between RRMS and SPMS groups (mean EDSS of RRMS = 2.2; mean EDSS of SPMS = 5.0). PBMCs were isolated by conventional Ficoll-Hypaque method from freshly drawn blood specimens. 2.2. Proliferative assays PBMC were cultured in triplicate at 1 105 cells per 200 Al well either alone or with anti-CD3 (1 Ag/ml; Sigma, St. Louis, MO), or PHA (5 Ag/ml; Sigma). Cultured PBMCs were pulsed with 1 ACi per well of tritiated methyl thymidine for the final 18 h of the 3-day culture
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in standard proliferative assays as described (Voskuhl et al., 1993). 2.3. Analysis of secreted cytokines by cytometric bead array PBMCs were cultured in 200 Al wells as above for 48 h with media and stimulated with anti-CD3 (1 Ag/ml; Sigma) or PHA (5 Ag/ml; Sigma). Supernatants were harvested and stored at 70 jC until cytokine testing was performed. IL-2, IL-4, IL-5, IL-10, TNF-a, and IFNg were detected simultaneously using the human Th1/Th2 cytokine cytometric bead array (CBA) kit (BD Biosciences Pharmingen, San Diego, CA). IL-12 p40 secretion from cell culture supernatants was measured by ELISA (R&D Systems, Minneapolis, MN). 2.4. Intracellular cytokine and surface marker staining PBMC were cultured for 24 h in 200 Al wells with stimulations as above. Brefeldin A was added during the last
Fig. 2. Decreased intracellular IL-10 and increased IL-12 p40 in SPMS. Intracellular cytokine levels (IL-5, IL-10, IL-12p40, TNF-a, and IFN-g) were measured in PBMC from RRMS, SPMS and ND patients following 24 h stimulation with (a) aCD3 and (b) PHA. (a) A trend toward decreased IL-10 production was observed in SPMS compared to RRMS 24 h after aCD3 stimulation ( p = 0.06). In contrast, IL-12p40 was increased in comparison with ND ( p = 0.04). (b) 24 h after stimulation with PHA, decreased IL-10 was observed in SPMS as compared to RRMS ( p = 0.001) and ND ( p = 0.006), and increased IL-12p40 were observed in SPMS as compared to RRMS ( p = 0.007) and ND ( p = 0.001). Data are represented as the total of cytokine positive cells after subtraction of staining observed in unstimulated PBMC. Bars represent intragroup mean. Asterisks: p < 0.05.
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5 h of stimulation. Cells were stained with FITC conjugated surface marker specific antibodies (CD4, CD8, CD19, CD64) at 4 jC, before being fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences Pharmingen). Cells were then stained with PE labeled antibodies specific for IL-5, IL-10, IL12p40, TNF-a, and IFN-g (?BD Biosciences Pharmingen?) at room temperature, before under-
going FACS analysis. Cells were also stained with isotype control antibodies to establish background staining and to set quadrants before calculation of the percentage of positive cells. Acquisition and analysis of % cytokine positive cells were performed on scatter gating (based on cell size and granularity) of both lymphocytes and monocytes (R1 + R2; Fig. 3).
Fig. 3. Representative data from PHA stimulated PBMC of an SPMS patient. IL-10 and IL-12p40 producing cell subsets were determined by double staining with cell surface antibodies (CD4 or CD64 FITC) and cytokine specific antibodies (IL-10 or IL-12p40 PE). Acquisition and analysis were performed by scatter gating (A) of lymphocytes (R1) and monocytes (R2) by cell size and granularity. Isotype controls were used for CD4+ T cells and CD64+ monocytes/ macrophages (B and C). IL-10 production was observed in both CD4+ T-cells and CD64+ monocytes while IL-12p40 expression was observed in CD64+ cells (G) but not in CD4+ T-cells (F).
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2.5. Statistical analysis The Student t-distribution was used to compare secreted cytokine levels (pg/ml) and % positive cells by intracellular cytokine staining between the RRMS, SPMS, and ND cohorts. Type I regression analysis was used to compare gadolinium enhancing lesion volumes, gadolinium enhancing lesion numbers, T2 lesion volumes and EDSS with secreted cytokine levels (pg/ml) and % positive cells by intracellular cytokine staining after aCD3 and PHA stimulation of PBMCs from SPMS patients.
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Table 1 Mean IL-10 and IL-12p40 production in CD4+ (%R1) and CD64+ (%R2) PBMC following PHA stimulation Mean frequency of cytokine producing cells
RRMS SPMS ND
CD4 + IL-10+%R1
CD64 + IL-10+%R2
CD64 + IL-12p40+%R2
10.4% (8.6 – 13.4%) 7.2% (4.5 – 9.2%)* 11.1% (6.5 – 14.3%)
9.8% (7.7 – 12.6%) 6.9% (3.5 – 9.8%)* 9.3% (7.8% – 14.1%)
13.6% (10.4 – 17.7%) 25.4% (18.1 – 39.1%)* 12.3% (7.64 – 16.32%)
* p < 0.02; Student’s t-test.
3. Results 3.1. Proliferative responses Lymphoproliferative responses to ex vivo stimulation with soluble aCD3 and PHA were assessed in 12 female MS patients (6 RRMS and 6 SPMS) and 10 normal donors. No significant difference in lymphoproliferative response to either aCD3 or PHA was observed between the RRMS, SPMS, or ND groups (data not shown). 3.2. Secreted cytokine differences Cytokine secretion (IL-2, IL-4, IL-5, IL-10, TNF-a, IFN-g) was measured from tissue culture supernatants by cytometric bead array. As demonstrated in Fig. 1a, a statistically significant decrease in IL-10 secretion was demonstrated in aCD3 stimulated PBMC of SPMS compared to those from both RRMS, and ND (Fig. 1a). In addition, IL-12p40 production was significantly increased in aCD3 stimulated PBMC of SPMS (Fig. 1a). No differences in IL-2, IL-4, IL-5, TNF-a, or IFN-g production were observed in PBMC from SPMS, RRMS, and ND stimulated with soluble aCD3. Significantly decreased levels of IL-10 and increased levels of IL-12p40 were also observed in culture supernatant from SPMS PBMC stimulated with PHA (Fig. 1b). 3.3. Intracellular cytokine differences Intracellular cytokine staining with cell surface marker specific antibodies was performed to confirm differences in cytokine production in supernatants between the SPMS, RRMS, and ND cohorts. As shown in Fig. 2a, intracellular cytokine staining of PBMC after stimulation with aCD3 confirmed increased production of IL-12p40 in this SPMS cohort. Decreased IL-10 production in SPMS as compared to RRMS was also observed. Again, no differences were observed in IL-5, TNF-a, or IFN-g production (Fig. 2a). The increased production of IL-12p40 and decreased production of IL-10 in response to PHA stimulation was confirmed in the SPMS cohort by intracellular cytokine analysis (Fig. 2b).
IL-10 production was observed in CD64+ macrophages and CD4+ T-cells, while IL-12p40 was detected primarily in CD64+ monocytes/macrophages in response to stimulation with both aCD3 and PHA in all subjects (Fig. 3). In addition to examining cytokine production in stimulated bulk PBMC (R1 + R2; Fig. 2), IL-10 and IL-12 production was analyzed in lymphocyte (R1, Fig. 3) and monocyte (R2; Fig. 3) enriched populations (Table 1). Intracellular IL-10 production was significantly decreased in CD4+ cells from PHA stimulated PBMC from SPMS compared to those from RRMS ( p = 0.01) and ND ( p = 0.004). IL-10 production was also significantly decreased in CD64+ cells from SPMS compared to RRMS ( p = 0.009) and ND ( p = 0.02). Moreover, IL-12p40 production was significantly increased in CD64+ cells from SPMS compared to both RRMS ( p = 0.006) and ND ( p = 0.001). An increase in IL-10 production in CD4+ and CD64+ cells and decrease in IL12p40 production in CD64+ cells was also observed in cells stimulated with aCD3 (not shown). 3.4. Cytokine levels and markers of clinical disease activity It was of interest to determine whether the increased IL12p40 production and decreased IL-10 production observed in SPMS correlated with clinical measures of disease activity, including EDSS and MRI measures (T2 or gadolinium enhancing lesions). No statistically significant correlation was demonstrated between cytokine levels and either EDSS or MRI measures in this relatively small cohort of SPMS patients.
4. Discussion The RRMS phase of the disease typically evolves into the SPMS phase, which is associated with neurologic deterioration and the accumulation of disability. However, the immunopathogenic changes that accompany the transition from the RRMS to SPMS phase are not well understood. To address this issue, we focused on cytokine production in female stable RRMS and SPMS patients and controls in response to ex vivo stimulation with aCD3 antibody and the
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mitogen PHA. Decreased IL-10 production was observed in SPMS compared to both RRMS and ND. Further, this decreased IL-10 production was accompanied by an increase in IL-12p40 in SPMS. These differences in cytokine production were observed in tissue culture supernatants and confirmed by intracellular cytokine staining. Previously, extensive work has characterized changes in cytokine production that precede or coincide with relapses in RRMS patients. More recent studies have begun to address whether cytokines differ in stable RRMS and SPMS patients. Our findings are consistent with an initial study demonstrating an increase in IL-12 secretion in SPMS as compared to RRMS and controls (Balashov et al., 1997). Also, our data at the protein level are consistent with data at the RNA level in a previous study in which MS patients had increased levels of IL-12p40 message and decreased IL-10 message in unstimulated PBMCs, with differences most pronounced in SPMS patients (van Boxel-Dezaire et al., 1999). From these reports and ours it cannot be known whether increased IL-12 induces decreased IL-10 or decreased IL-10 induces increased IL-12 or each occur independently in vivo. Further, since IL-12 and IL-23 share the IL-12p40 subunit (Cua et al., 2003), it is possible that an association of IL-23 with SPMS exists. Environmental influence or genetic background could each contribute to the evolution of the immune system from RRMS to SPMS. Regarding the environment, the transition to SPMS could be related to an increase in IL12 due to ongoing, repeated exposure to microbial products. Microbial products such as LPS, bacterial DNA, or oligonucleotides containing an unmethylated cytosine – guanine dinucleotide have been shown to promote encephalitogencity of myelin specific T cells in experimental autoimmune encephalomyelitis (EAE), with this action dependent on the ability of the microbial products to induce the production of IL-12 (Segal et al., 1997). The disease-promoting effects of IL-12 were antagonized by IL-10 which, in turn, was regulated by the endogenous production of IL-12 (Segal et al., 1998). This immunoregulatory circuit may play a critical role not only in EAE, but also in MS, and may provide a mechanism by which microbial triggers of the innate immune system could facilitate the transition from RRMS to SPMS. Genetic background is clearly important in MS susceptibility and is also likely to affect disease progression. Recently, an increased risk for a secondary progressive course was demonstrated in patients carrying at least one wild-type copy of the 1284A/C single-nucleotide polymorphism of the osteopontin gene (Caillier et al., 2003). Osteopontin is a newly described proinflammatory cytokine gene expressed abundantly within MS lesions (Chabas et al., 2001). Osteopontin has been shown to increase levels of IL-12 and decrease IL-10 (Ashkar et al., 2000). If levels of osteopontin were higher or functionally more potent in SPMS than in stable RRMS, then this could influence the disparities in cytokine production observed between dis-
ease types. While plasma osteopontin levels appeared no different between stable RRMS and SPMS (Vogt et al., 2003), this does not rule out differences in tissue expression or functional potency. Notably the above data each differ from a study showing an increase in IFN-g and IL-10 in SPMS with a defect in the ability of increased IL-10 to downregulate IFN-g and IL-12 in vitro (Balashov et al., 2000). Differences between reports may be due to differences in methodologies used in examining cytokine production. Alternatively, since gender differences in cytokine production have been shown (Pelfrey et al., 2002; Nguyen et al., 2003), differences may be related to the relative proportion of females versus males studied. Our study focused exclusively on females to avoid the confounding issue of gender. Collectively, these studies suggest that the immunopathogenesis of MS is influenced by both disease phenotype and gender. The elucidation of underlying differences in immune responsiveness in RRMS and SPMS are important in the understanding of the evolution of the immunopathogenesis of the disease and for the development of disease type specific treatments. Future studies including separate cohorts of male and female patients followed longitudinally during the transition from RRMS to SPMS with clinical, MRI and immune correlates are needed. Acknowledgements This work was supported by the National Institutes of Health grants AI50839, NS45443 (RRV); and the National Multiple Sclerosis Society grants RG3016 and RD3407 (RRV). Nancy Sicotte is Harry Weaver Neuroscience scholar of the National Multiple Sclerosis Society (JF2107). References Ashkar, S., Weber, G.F., Panoutsakopoulou, V., Sanchirico, M.E., Jansson, M., Zawaideh, S., Rittling, S.R., Denhardt, D.T., Glimcher, M.J., Cantor, H., 2000. Eta-1 (osteopontin): an early component of type-1 (cellmediated) immunity. Science 287, 860 – 864. Balashov, K.E., Smith, D.R., Khoury, S.J., Hafler, D.A., Weiner, H.L., 1997. Increased interleukin 12 production in progressive multiple sclerosis: induction by activated CD4+ T cells via CD40 ligand. Proc. Natl. Acad. Sci. U. S. A. 94, 599 – 603. Balashov, K.E., Comabella, M., Ohashi, T., Khoury, S.J., Weiner, H.L., 2000. Defective regulation of IFNgamma and IL-12 by endogenous IL-10 in progressive MS. Neurology 55, 192 – 198. Beck, J., Rondot, P., Catinot, L., Falcoff, E., Kirchner, H., Wietzerbin, J., 1988. Increased production of interferon gamma and tumor necrosis factor precedes clinical manifestation in multiple sclerosis: do cytokines trigger off exacerbations? Acta Neurol. Scand. 78, 318 – 323. Bjartmar, C., Wujek, J.R., Trapp, B.D., 2003. Axonal loss in the pathology of MS: consequences for understanding the progressive phase of the disease. J. Neurol. Sci. 206, 165 – 171. Caillier, S., Barcellos, L.F., Baranzini, S.E., Swerdlin, A., Lincoln, R.R., Steinman, L., Martin, E., Haines, J.L., Pericak-Vance, M., Hauser, S.L., Oksenberg, J.R., 2003. Osteopontin polymorphisms and disease course in multiple sclerosis. Genes Immun. 4, 312 – 315.
S.S. Soldan et al. / Journal of Neuroimmunology 146 (2004) 209–215 Chabas, D., Baranzini, S.E., Mitchell, D., Bernard, C.C., Rittling, S.R., Denhardt, D.T., Sobel, R.A., Lock, C., Karpuj, M., Pedotti, R., Heller, R., Oksenberg, J.R., Steinman, L., 2001. The influence of the proinflammatory cytokine, osteopontin, on autoimmune demyelinating disease. Science 294, 1731 – 1735. Cua, D.J., Sherlock, J., Chen, Y., Murphy, C.A., Joyce, B., Seymour, B., Lucian, L., To, W., Kwan, S., Churakova, T., Zurawski, S., Wiekowski, M., Lira, S.A., Gorman, D., Kastelein, R.A., Sedgwick, J.D., 2003. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421, 744 – 748. Duda, P.W., Schmied, M.C., Cook, S.L., Krieger, J.I., Hafler, D.A., 2000. Glatiramer acetate (Copaxone) induces degenerate, Th2-polarized immune responses in patients with multiple sclerosis. J. Clin. Invest. 105, 967 – 976. Duquette, P., Girard, M., 1993. Hormonal factors in susceptibility to multiple sclerosis. Curr. Opin. Neurol. Neurosurg. 6, 195 – 201. Duquette, P., Pleines, J., Girard, M., Charest, L., Senecal-Quevillon, M., Masse, C., 1992. The increased susceptibility of women to multiple sclerosis. Can. J. Neurol. Sci. 19, 466 – 471. Kim, S., Voskuhl, R.R., 1999. Decreased IL-12 production underlies the decreased ability of male lymph node cells to induce experimental autoimmune encephalomyelitis. J. Immunol. 162, 5561 – 5568. Lu, C.Z., Jensen, M.A., Arnason, B.G., 1993. Interferon gamma- and interleukin-4-secreting cells in multiple sclerosis. J. Neuroimmunol. 46, 123 – 128. Nguyen, L.T., Ramanathan, M., Weinstock-Guttman, B., Baier, M., Brownscheidle, C., Jacobs, L.D., 2003. Sex differences in in vitro pro-inflammatory cytokine production from peripheral blood of multiple sclerosis patients. J. Neurol. Sci. 209, 93 – 99. Pelfrey, C., Cotleur, A., Lee, J., Rudick, R., 2002. Sex differences in cytokine responses to myelin peptides in multiple sclerosis. J. Neuroimmunol. 130, 211. Rieckmann, P., Albrecht, M., Kitze, B., Weber, T., Tumani, H., Broocks, A., Luer, W., Helwig, A., Poser, S., 1995. Tumor necrosis factor-alpha messenger RNA expression in patients with relapsing – remitting multiple sclerosis is associated with disease activity. Ann. Neurol. 37, 82 – 88. Rudick, R.A., Ransohoff, R.M., Lee, J.C., Peppler, R., Yu, M.,
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Mathisen, P.M., Tuohy, V.K., 1998. In vivo effects of interferon beta-1a on immunosuppressive cytokines in multiple sclerosis. Neurology 50, 1294 – 1300 [Published erratum appears in Neurology 1998 Jul;51(1):332]. Segal, B.M., Klinman, D.M., Shevach, E.M., 1997. Microbial products induce autoimmune disease by an IL-12-dependent pathway. J. Immunol. 158, 5087 – 5090. Segal, B.M., Dwyer, B.K., Shevach, E.M., 1998. An interleukin (IL)-10/IL12 immunoregulatory circuit controls susceptibility to autoimmune disease. J. Exp. Med. 187, 537 – 546. Sinigaglia, F., D’Ambrosio, D., Rogge, L., 1999. Type I interferons and the Th1/Th2 paradigm. Dev. Comp. Immunol. 23, 657 – 663. Sorensen, T.L., Sellebjerg, F., 2001. Distinct chemokine receptor and cytokine expression profile in secondary progressive MS. Neurology 57, 1371 – 1376. Trapp, B.D., Peterson, J., Ransohoff, R.M., Rudick, R., Mork, S., Bo, L., 1998. Axonal transection in the lesions of multiple sclerosis. N. Engl. J. Med. 338, 278 – 285. Trapp, B.D., Ransohoff, R., Rudick, R., 1999. Axonal pathology in multiple sclerosis: relationship to neurologic disability. Curr. Opin. Neurol. 12, 295 – 302. van Boxel-Dezaire, A.H., Hoff, S.C., van Oosten, B.W., Verweij, C.L., Drager, A.M., Ader, H.J., van Houwelingen, J.C., Barkhof, F., Polman, C.H., Nagelkerken, L., 1999. Decreased interleukin-10 and increased interleukin-12p40 mRNA are associated with disease activity and characterize different disease stages in multiple sclerosis. Ann. Neurol. 45, 695 – 703. Vogt, M.H., Lopatinskaya, L., Smits, M., Polman, C.H., Nagelkerken, L., 2003. Elevated osteopontin levels in active relapsing – remitting multiple sclerosis. Ann. Neurol. 53, 819 – 822. Voskuhl, R.R., Martin, R., McFarland, H.F., 1993. A functional basis for the association of HLA class II genes and susceptibility to multiple sclerosis: cellular immune responses to myelin basic protein in a multiplex family. J. Neuroimmunol. 42, 199 – 207. Whitacre, C.C., Reingold, S.C., O’Looney, P.A., 1999. A gender gap in autoimmunity. Science 283, 1277 – 1278.
Short communication
Dysregulation of IL-10 and IL-12p40 in secondary progressive multiple sclerosis Samantha S. Soldan, Ana Isabel Alvarez Retuerto, Nancy L. Sicotte, Rhonda R. Voskuhl * Department of Neurology, Reed Neurological Research Center, University of California School of Medicine, 750 Westwood Plaza, Los Angeles, CA 90095, USA Received 28 July 2003; received in revised form 3 October 2003; accepted 3 October 2003
Abstract Multiple sclerosis (MS) is a putative T helper 1 (Th-1) mediated inflammatory disorder of the central nervous system, and levels of proinflammatory and anti-inflammatory cytokines have been found to correlate with changes in MS relapses. However, it is unclear if cytokine profiles differ between relapsing – remitting (RRMS) versus secondary progressive (SPMS) disease stages. Cytokine production (IL-2, IL-4, IL-5, IL-10, IL-12p40, TNF-a, and IFN-g) was assessed by cytometric bead array (CBA) and intracellular cytokine staining from aCD3 antibody and mitogen stimulated peripheral blood mononuclear cells (PBMCs) from female RRMS and SPMS patients, and healthy controls. Significantly increased production of IL-12p40 and decreased production of IL-10 were observed in SPMS patients. Differences in immune responsiveness in RRMS and SPMS are important in the understanding of the evolution of the immunopathogenesis of the disease and for the development of disease type specific treatments. D 2003 Elsevier B.V. All rights reserved. Keywords: Multiple sclerosis; Cytokines; Th1/Th2
1. Introduction Multiple sclerosis is the most prevalent demyelinating disease of young adults with a clear gender bias involving an increased incidence in females (Duquette et al., 1992; Duquette and Girard, 1993; Whitacre et al., 1999). The most common form of MS is the relapsing – remitting MS (RRMS) phenotype that is characterized by disease exacerbations where new symptoms appear or existing symptoms become more severe. Most individuals who are initially diagnosed with RRMS will develop secondary progressive MS (SPMS) marked by the gradual accumulation of progressive disability. It is widely believed that T helper 1 (Th-1) mediated responses are involved in the pathogenesis of multiple sclerosis (MS) (Lu et al., 1993; Rieckmann et al., 1995; Beck et al., 1988). This is based on the association of MS with genes involved with the immune system, the immunopathology of the disease, the clinical response of MS
* Corresponding author. Tel.: +1-310-206-4636; fax: +1-310-2069801. E-mail address: [email protected] (R.R. Voskuhl). 0165-5728/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2003.10.033
patients to immunomodulatory and immunosuppressive treatments, and similarities with experimental immune-mediated demyelinating diseases in animals. According to the Th-1/Th-2 paradigm, Th-1 lymphocytes secrete proinflammatory cytokines (e.g. IFNg, TNFa), while Th-2 lymphocytes produce anti-inflammatory cytokines (e.g. IL-4, IL-5, IL-10) that promote humoral responses and confer protection from Th-1 mediated autoimmunity. While the counterregulatory roles of Th-1 and Th-2 cytokines are wellestablished in murine systems, this dichotomy is more ambiguous in humans (Sinigaglia et al., 1999). Nevertheless, the Th-1/Th-2 paradigm has been useful in understanding the mechanism of immunomodulatory therapies for MS (Duda et al., 2000; Rudick et al., 1998). As compared to RRMS, SPMS is characterized by fewer enhancing lesions on brain MRI and greater axonal degeneration (Trapp et al., 1998; Bjartmar et al., 2003; Trapp et al., 1999). Unfortunately, when patients transition from RRMS to SPMS, their disease often becomes refractory to immunomodulatory therapies. The disparate therapeutic effects of immunomodulatory therapy in these disease phases may reflect differences in the relative contribution of axonal pathology to disease course and/or differences in the immunopathogenesis of these disease phases. Some
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differences in immune dysregulation between RRMS and SPMS have been described (Balashov et al., 1997, 2000; Pelfrey et al., 2002; Sorensen and Sellebjerg, 2001; van Boxel-Dezaire et al., 1999). While recent studies have reported distinctly altered cytokine profiles in RRMS and SPMS, there is no consensus between these studies regarding the relationship between cytokine profiles and disease phenotype (Balashov et al., 2000; van Boxel-Dezaire et al., 1999; Pelfrey et al., 2002). In this study, cytokine production was determined in response to ex vivo stimulation. We focused exclusively on female subjects to assess differences in cytokine profiles between disease phases while eliminat-
ing the confounding effects of gender (Kim and Voskuhl, 1999; Nguyen et al., 2003; Pelfrey et al., 2002).
2. Materials and methods 2.1. Patient and control samples Peripheral blood mononuclear cells (PBMCs) were obtained from 12 female patients with clinically definite MS (6 RR and 6 SP) and 10 female, age-matched healthy controls (ND). MS patients and ND were screened by history
Fig. 1. Decreased IL-10 and increased IL-12p40 secretion in SPMS. Secreted cytokine levels (IL-2, IL-4, IL-5, IL-10, IL-12 p40, TNF-a, and IFN-g) were measured in tissue culture supernatant 48 h after stimulation with (a) soluble aCD3 and (b) PHA. (a) Decreased IL-10 production was observed in aCD3 stimulated SPMS PBMC compared to both RRMS ( p = 0.003) and ND ( p = 0.002). In addition, IL-12p40 production was increased SPMS compared to RRMS ( p = 0.003) and ND ( p = 0.003). (b) IL-10 production was also significantly decreased in SPMS PBMC stimulated with PHA. This decrease in IL-10 was observed in comparison to PBMC from RRMS ( p < 0.01) and ND ( p < 0.01). IL-12 was significantly increased in SPMS PBMC stimulated with PHA as compared to RRMS ( p = 0.028) and ND ( p = 0.04). The range of detection was 20 – 5000 pg/ml for each cytokine measured by CBA and 20 – 2000 for IL12p40. Bars represent intragroup mean. Asterisks: p < 0.05.
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and serologic studies and did not present evidence of chronic infectious, neoplastic, or other autoimmune diseases. MS patients had not received steroid treatment for at least 3 months or IFN-h or glatiramer acetate for 6 months prior. The mean age of the MS patients was 44 years (range, 28 –50 years) while the mean age of the ND group was 38 years (range, 21 –58). The mean Expanded Disability Status Scale (EDSS) score was 3.3 (range, 1.0 – 6.5) with clear differences between RRMS and SPMS groups (mean EDSS of RRMS = 2.2; mean EDSS of SPMS = 5.0). PBMCs were isolated by conventional Ficoll-Hypaque method from freshly drawn blood specimens. 2.2. Proliferative assays PBMC were cultured in triplicate at 1 105 cells per 200 Al well either alone or with anti-CD3 (1 Ag/ml; Sigma, St. Louis, MO), or PHA (5 Ag/ml; Sigma). Cultured PBMCs were pulsed with 1 ACi per well of tritiated methyl thymidine for the final 18 h of the 3-day culture
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in standard proliferative assays as described (Voskuhl et al., 1993). 2.3. Analysis of secreted cytokines by cytometric bead array PBMCs were cultured in 200 Al wells as above for 48 h with media and stimulated with anti-CD3 (1 Ag/ml; Sigma) or PHA (5 Ag/ml; Sigma). Supernatants were harvested and stored at 70 jC until cytokine testing was performed. IL-2, IL-4, IL-5, IL-10, TNF-a, and IFNg were detected simultaneously using the human Th1/Th2 cytokine cytometric bead array (CBA) kit (BD Biosciences Pharmingen, San Diego, CA). IL-12 p40 secretion from cell culture supernatants was measured by ELISA (R&D Systems, Minneapolis, MN). 2.4. Intracellular cytokine and surface marker staining PBMC were cultured for 24 h in 200 Al wells with stimulations as above. Brefeldin A was added during the last
Fig. 2. Decreased intracellular IL-10 and increased IL-12 p40 in SPMS. Intracellular cytokine levels (IL-5, IL-10, IL-12p40, TNF-a, and IFN-g) were measured in PBMC from RRMS, SPMS and ND patients following 24 h stimulation with (a) aCD3 and (b) PHA. (a) A trend toward decreased IL-10 production was observed in SPMS compared to RRMS 24 h after aCD3 stimulation ( p = 0.06). In contrast, IL-12p40 was increased in comparison with ND ( p = 0.04). (b) 24 h after stimulation with PHA, decreased IL-10 was observed in SPMS as compared to RRMS ( p = 0.001) and ND ( p = 0.006), and increased IL-12p40 were observed in SPMS as compared to RRMS ( p = 0.007) and ND ( p = 0.001). Data are represented as the total of cytokine positive cells after subtraction of staining observed in unstimulated PBMC. Bars represent intragroup mean. Asterisks: p < 0.05.
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5 h of stimulation. Cells were stained with FITC conjugated surface marker specific antibodies (CD4, CD8, CD19, CD64) at 4 jC, before being fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences Pharmingen). Cells were then stained with PE labeled antibodies specific for IL-5, IL-10, IL12p40, TNF-a, and IFN-g (?BD Biosciences Pharmingen?) at room temperature, before under-
going FACS analysis. Cells were also stained with isotype control antibodies to establish background staining and to set quadrants before calculation of the percentage of positive cells. Acquisition and analysis of % cytokine positive cells were performed on scatter gating (based on cell size and granularity) of both lymphocytes and monocytes (R1 + R2; Fig. 3).
Fig. 3. Representative data from PHA stimulated PBMC of an SPMS patient. IL-10 and IL-12p40 producing cell subsets were determined by double staining with cell surface antibodies (CD4 or CD64 FITC) and cytokine specific antibodies (IL-10 or IL-12p40 PE). Acquisition and analysis were performed by scatter gating (A) of lymphocytes (R1) and monocytes (R2) by cell size and granularity. Isotype controls were used for CD4+ T cells and CD64+ monocytes/ macrophages (B and C). IL-10 production was observed in both CD4+ T-cells and CD64+ monocytes while IL-12p40 expression was observed in CD64+ cells (G) but not in CD4+ T-cells (F).
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2.5. Statistical analysis The Student t-distribution was used to compare secreted cytokine levels (pg/ml) and % positive cells by intracellular cytokine staining between the RRMS, SPMS, and ND cohorts. Type I regression analysis was used to compare gadolinium enhancing lesion volumes, gadolinium enhancing lesion numbers, T2 lesion volumes and EDSS with secreted cytokine levels (pg/ml) and % positive cells by intracellular cytokine staining after aCD3 and PHA stimulation of PBMCs from SPMS patients.
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Table 1 Mean IL-10 and IL-12p40 production in CD4+ (%R1) and CD64+ (%R2) PBMC following PHA stimulation Mean frequency of cytokine producing cells
RRMS SPMS ND
CD4 + IL-10+%R1
CD64 + IL-10+%R2
CD64 + IL-12p40+%R2
10.4% (8.6 – 13.4%) 7.2% (4.5 – 9.2%)* 11.1% (6.5 – 14.3%)
9.8% (7.7 – 12.6%) 6.9% (3.5 – 9.8%)* 9.3% (7.8% – 14.1%)
13.6% (10.4 – 17.7%) 25.4% (18.1 – 39.1%)* 12.3% (7.64 – 16.32%)
* p < 0.02; Student’s t-test.
3. Results 3.1. Proliferative responses Lymphoproliferative responses to ex vivo stimulation with soluble aCD3 and PHA were assessed in 12 female MS patients (6 RRMS and 6 SPMS) and 10 normal donors. No significant difference in lymphoproliferative response to either aCD3 or PHA was observed between the RRMS, SPMS, or ND groups (data not shown). 3.2. Secreted cytokine differences Cytokine secretion (IL-2, IL-4, IL-5, IL-10, TNF-a, IFN-g) was measured from tissue culture supernatants by cytometric bead array. As demonstrated in Fig. 1a, a statistically significant decrease in IL-10 secretion was demonstrated in aCD3 stimulated PBMC of SPMS compared to those from both RRMS, and ND (Fig. 1a). In addition, IL-12p40 production was significantly increased in aCD3 stimulated PBMC of SPMS (Fig. 1a). No differences in IL-2, IL-4, IL-5, TNF-a, or IFN-g production were observed in PBMC from SPMS, RRMS, and ND stimulated with soluble aCD3. Significantly decreased levels of IL-10 and increased levels of IL-12p40 were also observed in culture supernatant from SPMS PBMC stimulated with PHA (Fig. 1b). 3.3. Intracellular cytokine differences Intracellular cytokine staining with cell surface marker specific antibodies was performed to confirm differences in cytokine production in supernatants between the SPMS, RRMS, and ND cohorts. As shown in Fig. 2a, intracellular cytokine staining of PBMC after stimulation with aCD3 confirmed increased production of IL-12p40 in this SPMS cohort. Decreased IL-10 production in SPMS as compared to RRMS was also observed. Again, no differences were observed in IL-5, TNF-a, or IFN-g production (Fig. 2a). The increased production of IL-12p40 and decreased production of IL-10 in response to PHA stimulation was confirmed in the SPMS cohort by intracellular cytokine analysis (Fig. 2b).
IL-10 production was observed in CD64+ macrophages and CD4+ T-cells, while IL-12p40 was detected primarily in CD64+ monocytes/macrophages in response to stimulation with both aCD3 and PHA in all subjects (Fig. 3). In addition to examining cytokine production in stimulated bulk PBMC (R1 + R2; Fig. 2), IL-10 and IL-12 production was analyzed in lymphocyte (R1, Fig. 3) and monocyte (R2; Fig. 3) enriched populations (Table 1). Intracellular IL-10 production was significantly decreased in CD4+ cells from PHA stimulated PBMC from SPMS compared to those from RRMS ( p = 0.01) and ND ( p = 0.004). IL-10 production was also significantly decreased in CD64+ cells from SPMS compared to RRMS ( p = 0.009) and ND ( p = 0.02). Moreover, IL-12p40 production was significantly increased in CD64+ cells from SPMS compared to both RRMS ( p = 0.006) and ND ( p = 0.001). An increase in IL-10 production in CD4+ and CD64+ cells and decrease in IL12p40 production in CD64+ cells was also observed in cells stimulated with aCD3 (not shown). 3.4. Cytokine levels and markers of clinical disease activity It was of interest to determine whether the increased IL12p40 production and decreased IL-10 production observed in SPMS correlated with clinical measures of disease activity, including EDSS and MRI measures (T2 or gadolinium enhancing lesions). No statistically significant correlation was demonstrated between cytokine levels and either EDSS or MRI measures in this relatively small cohort of SPMS patients.
4. Discussion The RRMS phase of the disease typically evolves into the SPMS phase, which is associated with neurologic deterioration and the accumulation of disability. However, the immunopathogenic changes that accompany the transition from the RRMS to SPMS phase are not well understood. To address this issue, we focused on cytokine production in female stable RRMS and SPMS patients and controls in response to ex vivo stimulation with aCD3 antibody and the
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mitogen PHA. Decreased IL-10 production was observed in SPMS compared to both RRMS and ND. Further, this decreased IL-10 production was accompanied by an increase in IL-12p40 in SPMS. These differences in cytokine production were observed in tissue culture supernatants and confirmed by intracellular cytokine staining. Previously, extensive work has characterized changes in cytokine production that precede or coincide with relapses in RRMS patients. More recent studies have begun to address whether cytokines differ in stable RRMS and SPMS patients. Our findings are consistent with an initial study demonstrating an increase in IL-12 secretion in SPMS as compared to RRMS and controls (Balashov et al., 1997). Also, our data at the protein level are consistent with data at the RNA level in a previous study in which MS patients had increased levels of IL-12p40 message and decreased IL-10 message in unstimulated PBMCs, with differences most pronounced in SPMS patients (van Boxel-Dezaire et al., 1999). From these reports and ours it cannot be known whether increased IL-12 induces decreased IL-10 or decreased IL-10 induces increased IL-12 or each occur independently in vivo. Further, since IL-12 and IL-23 share the IL-12p40 subunit (Cua et al., 2003), it is possible that an association of IL-23 with SPMS exists. Environmental influence or genetic background could each contribute to the evolution of the immune system from RRMS to SPMS. Regarding the environment, the transition to SPMS could be related to an increase in IL12 due to ongoing, repeated exposure to microbial products. Microbial products such as LPS, bacterial DNA, or oligonucleotides containing an unmethylated cytosine – guanine dinucleotide have been shown to promote encephalitogencity of myelin specific T cells in experimental autoimmune encephalomyelitis (EAE), with this action dependent on the ability of the microbial products to induce the production of IL-12 (Segal et al., 1997). The disease-promoting effects of IL-12 were antagonized by IL-10 which, in turn, was regulated by the endogenous production of IL-12 (Segal et al., 1998). This immunoregulatory circuit may play a critical role not only in EAE, but also in MS, and may provide a mechanism by which microbial triggers of the innate immune system could facilitate the transition from RRMS to SPMS. Genetic background is clearly important in MS susceptibility and is also likely to affect disease progression. Recently, an increased risk for a secondary progressive course was demonstrated in patients carrying at least one wild-type copy of the 1284A/C single-nucleotide polymorphism of the osteopontin gene (Caillier et al., 2003). Osteopontin is a newly described proinflammatory cytokine gene expressed abundantly within MS lesions (Chabas et al., 2001). Osteopontin has been shown to increase levels of IL-12 and decrease IL-10 (Ashkar et al., 2000). If levels of osteopontin were higher or functionally more potent in SPMS than in stable RRMS, then this could influence the disparities in cytokine production observed between dis-
ease types. While plasma osteopontin levels appeared no different between stable RRMS and SPMS (Vogt et al., 2003), this does not rule out differences in tissue expression or functional potency. Notably the above data each differ from a study showing an increase in IFN-g and IL-10 in SPMS with a defect in the ability of increased IL-10 to downregulate IFN-g and IL-12 in vitro (Balashov et al., 2000). Differences between reports may be due to differences in methodologies used in examining cytokine production. Alternatively, since gender differences in cytokine production have been shown (Pelfrey et al., 2002; Nguyen et al., 2003), differences may be related to the relative proportion of females versus males studied. Our study focused exclusively on females to avoid the confounding issue of gender. Collectively, these studies suggest that the immunopathogenesis of MS is influenced by both disease phenotype and gender. The elucidation of underlying differences in immune responsiveness in RRMS and SPMS are important in the understanding of the evolution of the immunopathogenesis of the disease and for the development of disease type specific treatments. Future studies including separate cohorts of male and female patients followed longitudinally during the transition from RRMS to SPMS with clinical, MRI and immune correlates are needed. Acknowledgements This work was supported by the National Institutes of Health grants AI50839, NS45443 (RRV); and the National Multiple Sclerosis Society grants RG3016 and RD3407 (RRV). Nancy Sicotte is Harry Weaver Neuroscience scholar of the National Multiple Sclerosis Society (JF2107). References Ashkar, S., Weber, G.F., Panoutsakopoulou, V., Sanchirico, M.E., Jansson, M., Zawaideh, S., Rittling, S.R., Denhardt, D.T., Glimcher, M.J., Cantor, H., 2000. Eta-1 (osteopontin): an early component of type-1 (cellmediated) immunity. Science 287, 860 – 864. Balashov, K.E., Smith, D.R., Khoury, S.J., Hafler, D.A., Weiner, H.L., 1997. Increased interleukin 12 production in progressive multiple sclerosis: induction by activated CD4+ T cells via CD40 ligand. Proc. Natl. Acad. Sci. U. S. A. 94, 599 – 603. Balashov, K.E., Comabella, M., Ohashi, T., Khoury, S.J., Weiner, H.L., 2000. Defective regulation of IFNgamma and IL-12 by endogenous IL-10 in progressive MS. Neurology 55, 192 – 198. Beck, J., Rondot, P., Catinot, L., Falcoff, E., Kirchner, H., Wietzerbin, J., 1988. Increased production of interferon gamma and tumor necrosis factor precedes clinical manifestation in multiple sclerosis: do cytokines trigger off exacerbations? Acta Neurol. Scand. 78, 318 – 323. Bjartmar, C., Wujek, J.R., Trapp, B.D., 2003. Axonal loss in the pathology of MS: consequences for understanding the progressive phase of the disease. J. Neurol. Sci. 206, 165 – 171. Caillier, S., Barcellos, L.F., Baranzini, S.E., Swerdlin, A., Lincoln, R.R., Steinman, L., Martin, E., Haines, J.L., Pericak-Vance, M., Hauser, S.L., Oksenberg, J.R., 2003. Osteopontin polymorphisms and disease course in multiple sclerosis. Genes Immun. 4, 312 – 315.
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