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Increased IL-13-producing T cells in ALS: Positive correlations with disease severity and progression rate
Journal of Neuroimmunology 182 (2007) 232 – 235 www.elsevier.com/locate/jneuroim
Short communication
Increased IL-13-producing T cells in ALS: Positive correlations with disease severity and progression rate Nan Shi 1 , Yuji Kawano 1 , Takahisa Tateishi, Hitoshi Kikuchi, Manabu Osoegawa, Yasumasa Ohyagi, Jun-ichi Kira ⁎ Department of Neurology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan Received 22 June 2006; received in revised form 23 September 2006; accepted 2 October 2006
Abstract We measured the intracellular productions of IFNγ, IL-2, IL-4, IL-13 and TNFα in peripheral blood CD4+ and CD8+ T cells from 21 amyotrophic lateral sclerosis (ALS) patients, 14 disease controls (DC) with spinocerebellar degeneration and 16 healthy controls (HC). Only the percentages of CD4+IL-13+ and CD8+IL-13+ T cells were significantly higher in ALS patients than in DC and HC. The CD4+IL-13+ T cell percentages showed a significant negative correlation with the revised ALS functional rating scale scores and significant positive correlation with the disease progression rate, suggesting that IL-13 contributes to ALS. © 2006 Elsevier B.V. All rights reserved. Keywords: Amyotrophic lateral sclerosis; Interleukin-13; Neuroinflammation; CD4+ T cell; CD8+ T cell
1. Introduction
2. Subjects and methods
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that primarily affects the upper and lower motor neurons. Although its etiology remains to be elucidated, increasing evidence suggests that inflammatory processes may contribute to motor neuron damage in ALS. Specifically, proinflammatory cytokines, such as macrophage chemoattractant protein (MCP)-1 (Henkel et al., 2004; Tanaka et al., 2006), interleukin (IL)-5 (Tanaka et al., 2006), IL-6 (Sekizawa et al., 1998) and tumor necrosis factor-α (TNF-α) (Poloni et al., 2000), are increased in cerebrospinal fluid (CSF) and sera. Moreover, ALS spinal cord shows activation of microglia together with upregulation of MCP-1 mRNA (Henkel et al., 2004). These findings prompted us to study the intracellular cytokine productions of T helper type 1 (Th1) and Th2 cytokines in ALS patients.
2.1. Subjects
⁎ Corresponding author. Tel.: +81 92 642 5340; fax: +81 92 642 5352. E-mail address: [email protected] (J. Kira). 1 These authors contributed equally to this work. 0165-5728/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2006.10.001
A total of 21 Japanese patients (11 men, 10 women; mean age ± SD: 55 ± 15 years) with clinically definite ALS based on the El Escorial criteria (Brooks, 1994) who were not taking any drugs, 14 disease control patients with spinocerebellar degeneration (9 men, 5 women; mean age ± S.D.: 60 ± 14 years) and 16 healthy controls (12 men, 4 women; mean age ± S.D.: 44 ± 9 years) were enrolled in this study. At the time of blood sampling, none of the subjects were experiencing acute infections or inflammatory diseases. The disease duration of ALS was 24.8 ± 17.5 months. For the serum IgE assay, blood samples collected from the same 21 Japanese ALS patients as those enrolled for the intracellular cytokine assay and 59 healthy controls (30 men, 29 women; mean age ± S.D.: 47 ± 6 years) were evaluated. We evaluated the disabilities of the patients using the Revised ALS Functional Rating Scale (ALSFRS-R) (Cedarbaum et al., 1999), and found that their ALSFRS-R score was 34.6± 11.21 (mean ± S.D.; range: 0-48). We calculated the disease progression rate (DPR) as follows: DPR = (48− ALSFRS-
N. Shi et al. / Journal of Neuroimmunology 182 (2007) 232–235
R) / disease duration (months) (Tanaka et al., 2006). The DPR of the ALS patients was 0.82± 0.71 (mean± S.D.; range: 0.03–2.30). 2.2. Flow cytometry for intracellular cytokines The intracellular cytokine productions were studied by flow cytometry as described previously (Ochi et al., 2002). Briefly, peripheral blood mononuclear cells were collected from the subjects and stimulated with 25 ng/ml phorbol 12myristate 13-acetate (Sigma, St. Louis, MO) and 1 μg/ml of ionomycin (Sigma) in the presence of 10 μg/ml brefeldin A (Sigma) for 4 h at 37.5 °C. After washing with phosphatebuffered saline containing 0.1% bovine serum albumin (0.1% BSA-PBS), antibodies against cell surface markers were added for 15 min at room temperature in the dark. The cells were washed twice, permeabilized with FACS permeabilizing solution (Becton Dickinson, San Jose, CA) and incubated with antibodies against human cytokines or isotype-matched controls for 30 min. Finally, the cells were washed with 0.1% BSA-PBS and analyzed using an Epics XL System II (Coulter, Hialeah, FL). The monoclonal antibodies used were as follows: PC5-conjugated anti-CD4 (13B8.2; Becton Dickinson, San Jose, CA), PC5-conjugated anti-CD8 (B9.11; Becton Dickinson), FITC-conjugated anti-CD8 (B9.11; Beckman Coulter, Miami, FL), FITC-conjugated anti-IFNγ (25723.11; Becton Dickinson), PE-conjugated anti-IL-4 (3010.211; Becton Dickinson), PE-conjugated anti-TNF-α (6401.1111; Becton Dickinson), FITC-conjugated anti-IL-2 (5344.111; Becton Dickinson) and PE-conjugated anti-IL-13 (JES10-5A2; PharMingen, San Diego, CA). The percentages of cytokine-positive CD4+ and CD8+ cells were determined as the % cytokine-positive CD4+ population/total CD4+ population and % cytokine-positive CD8+ population/total CD8+ population, respectively.
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2.4. Statistical analysis Statistical analyses for comparisons of cell percentages were initially performed using the Kruskal–Wallis H test. When statistical significance was found, the Mann–Whitney U test was used to determine the statistical differences among the groups. Uncorrelated P values (Puncorr) were corrected by multiplying them by the number of comparisons (Bonferroni–Dunn's correction) to calculate corrected P values ( Pcorr). A Pcorr value of less than 0.05 was considered significant. Statistical analyses for comparisons of serum IgE concentrations were performed using the Mann–Whitney U test. The ALSFRS-R scores and IL-13-positive cell percentages among the CD4+ T cells showed normal distributions. However, the DPR and IL-13-positive cell percentages among the CD8+ T cells were not normally distributed. Therefore, the correlation between the cytokine-positive cell percentages in CD4+ T cells and ALSFRS-R scores was analyzed by Pearson's correlation test, while other correlations were analyzed by Spearman's rank correlation test. 3. Results 3.1. Intracellular cytokines in CD4+ T cells Among the cytokines examined, only the percentage of CD4+IL-13+ T cells was significantly higher in ALS patients than in healthy controls (Pcorr = 0.039) and disease controls (Pcorr = 0.024) (Table 1). The other percentages, including CD4+IL-4−IFNγ+, CD4+IL-4+IFNγ−, CD4+IL-2−TNFα+, CD4+IL-2+TNFα− and CD4+IL-2+TNFα+T cells, and the ratio of CD4+IL-4−IFNγ+T cells to CD4+IL-4+IFNγ−T cells (intracellular IFNγ/IL-4 ratio) did not differ significantly among the ALS patients, healthy controls and disease controls. 3.2. Intracellular cytokines in CD8+ T cells
2.3. Determination of total serum IgE Serum total IgE was measured using an enzyme-linked immunosorbent assay (ELISA) as described previously (Kira et al., 1997).
Similarly, only the percentage of CD8+IL-13+ T cells was significantly higher in ALS patients than in healthy controls (Pcorr = 0.006) (Table 1). The percentage of CD8+IL-13+ T cells was also higher in ALS patients than in disease controls,
Table 1 Intracellular cytokine productions in peripheral blood CD4+ and CD8+ T cells IL-4-IFN-γ+
IL-4+IFN-γ-
IL-4+IFN-γ+
CD4+T cells HC (n = 16) DC (n = 14) ALS (n = 21)
22.13 ± 7.16 25.59 ± 8.95 20.72 ± 6.23
1.24 ± 0.46 1.02 ± 0.6 1.46 ± 0.88
0.55 ± 0.46 0.22 ± 0.23 0.30 ± 0.24
CD8+T cells HC (n = 16) DC (n = 14) ALS (n = 21)
55.91 ± 16.48 64.24 ± 18.12 57.59 ± 17.75
0.91 ± 0.66 1.65 ± 2.24 1.82 ± 2.56
0.33 ± 0.33 0.34 ± 0.20 0.39 ± 0.37
IFN-γ+/IL-4+
IL-13+
IL-2-TNF-α+
IL-2+TNF-α-
IL-2+TNF-α+
20.16 ± 13.82 40.17 ± 53.55 23.76 ± 26.29
4.21 ± 2.58 3.54 ± 1.73 6.96 ± 3.97 a
20.38 ± 6.30 22.24 ± 9.99 17.97 ± 5.20
9.44 ± 3.56 12.74 ± 6.7 9.54 ± 3.85
31.03 ± 11.47 33.82 ± 8.21 32.80 ± 11.88
176.88 ± 293.80 275.0 ± 489.15 124.32 ± 143.93
2.85 ± 1.66 4.64 ± 6.19 6.94 ± 5.67 b
32.67 ± 11.47 36.89 ± 14.66 37.26 ± 17.95
7.44 ± 4.76 7.31 ± 4.70 5.05 ± 3.91
16.68 ± 9.36 22.67 ± 9.78 18.02 ± 11.46
ALS, amyotrophic lateral sclerosis patients; HC, healthy controls; DC, disease controls. a Significant difference vs. HC ( Pcorr b 0.05) or DC ( Pcorr b 0.05). b Significant difference vs. HC ( Pcorr b 0.05).
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N. Shi et al. / Journal of Neuroimmunology 182 (2007) 232–235
Fig. 1. Correlations between the peripheral blood IL-13+ cell percentages and the Revised ALS Functional Rating Scale (ALSFRS-R) scores (A and B) and between the peripheral blood IL-13+ cell percentages and the disease progression rate (C and D).
but the difference did not reach statistical significance (Pcorr N 0.1). 3.3. Total serum IgE concentration The mean serum IgE concentration was higher in ALS patients (mean ± S.D.: 181 ± 242 IU/ml) than in healthy controls (mean ± S.D.: 117 ± 148 IU/ml), but the difference did not reach statistical significance (P N 0.05) due to the small sample numbers. 3.4. Correlations between the ALSFRS-R score and IL-13 production, and between the DPR and IL-13 production The IL-13-positive cell percentages showed a significant negative correlation with the ALSFRS-R scores in CD4+ T cells (r = − 0.497, p = 0.022), but not in CD8+ T cells (Fig. 1). In addition, the IL-13-positive cell percentages showed a significant positive correlation with the DPR in CD4+ T cells (r = 0.496, p = 0.027), but not in CD8+ T cells (Fig. 1). 4. Discussion In the present study, we have revealed for the first time that IL-13 production is upregulated in peripheral blood CD4+ and CD8+ T cells in ALS and that the number of IL13-producing CD4+ T cells is significantly associated with the disease severity and progression rate.
Although no statistically significant difference in the serum IgE concentrations was observed between ALS patients and healthy controls due to the small sample numbers, the mean serum IgE concentration was higher in ALS patients than in healthy controls. The percentage of CD4+IL13+ T cells in ALS patients was as high as that seen in multiple sclerosis patients at relapse, in which we previously reported its upregulation (Ochi et al., 2002). Moreover, IL-13 production was positively correlated with the DPR. These findings suggest that upregulation of IL-13 production in ALS patients has some biological relevance and that IL-13 could be involved in the neurodegeneration observed in ALS. We detected no differences in other cytokine productions. These findings suggest that systemic immune activation, especially Th1 type immune activation, is not involved in ALS. Among the Th2 cytokines examined, IL-13 was upregulated in the present study, whereas IL-4 was not. Accumulating evidence has indicated that each Th2 cytokine has its own distinct regulation system. For example, c-Maf is essential for the expression of IL-4, but not IL-13 (Kim et al., 1999). On the contrary, IL-13 is regulated by GATA-3, while IL-4 is not (Zhu et al., 2004). Therefore, the isolated IL-13 upregulation seen in ALS is probably based on differences among the regulation systems of Th2 cytokines. Such isolated IL-13 upregulation was also observed in nephrotic syndrome at the relapse phase (Yap et al., 1999), where expression of IL-13, but not IL-2, IL-4 or INF-γ, was
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increased in T cells and considered to contribute to disease exacerbation through immunomodulatory effects on macrophages and B cells. IL-13 drives humoral immunity and potentiates IgE synthesis. Humoral immunity is thought to be involved in ALS, since IgG is deposited in the spinal cord and motor cortex in ALS (Engelhardt and Appel, 1990) and autoantibodies to calcium channels are present (Offen et al., 1998). In addition, the incidence of history of past and concomitant asthma is significantly increased in patients with motor neuron diseases in Japan, as evaluated in a largescale prospective study (Kira et al., 2002). Thus, the increased number of IL-13-producing T cells in ALS may be related to such abnormalities in humoral immunity. On the other hand, IL-13 enhances MCP-1 expression in monocytes/macrophages (Szczepanik et al., 2001). MCP-1 attracts monocytes/macrophages and CCR2-bearing Th2 cells and activates monocytes and microglia, thereby leading to the production of proinflammatory cytokines and nitric oxide. Furthermore, MCP-1 in CSF is positively correlated with disease severity (Tanaka et al., 2006). Therefore, IL-13 may aggravate ALS through MCP-1 induction and subsequent glial inflammation. CD4+ and CD8+ T cells are known to be present in the ALS spinal cord (Kawamata et al., 1992). IL-13 upregulates the expression of vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells, which plays a crucial role in mediating the adhesion and migration of inflammatory cells in the central nervous system (CNS). Moreover, IL-13 enhances the expression of MHC class II molecules in monocytes/macrophages (Cash et al., 1994), thereby potentiating the capacity of these cells to present relevant antigens. IL-13 may therefore facilitate the CNS inflammatory cascade in ALS. Our present results suggest that IL-13 contributes to the aggravation of ALS, probably through potentiation of inflammatory components of the disease. Acknowledgements This work was supported in part by grants from the Ministry of Education, Culture, Science and Technology of Japan, the Neuroimmunological Disease Research Committee, the Ministry of Health, Labor and Welfare of Japan and the Japan ALS Association. References Brooks, B.R., 1994. El Escorial World Federation of Neurology criteria for the diagnosis of amyotrophic lateral screrosis. Subcommittee on Motor Neuron Diseases/Amyotrophic Lateral Sclerosis of the World Federation of Neurology Research Group on Neuromuscular Diseases and the El Escorial “Clinical limits of amyotrophic lateral screlosis” workshop contributors. J. Neurol. Sci. 124, 96–107 (Suppl).
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Cash, E., Minty, A., Ferrare, O., Caput, D., Fradelizi, D., Rotto, O., 1994. Macrophage-inactivating IL-13 suppresses experimental autoimmune encephalomyelitis in rats. J. Immunol. 153, 4258–4267. Cedarbaum, J.M., Stambler, N., Malta, E., Fuller, C., Hilt, D., Thurmond, B., Nakanishi, A., 1999. The ALSFRS-R: A revised ALS functional rating scale that incorporates assessments of respiratory function. J. Neurol. Sci. 169, 13–21. Engelhardt, J.I., Appel, S.H., 1990. IgG reactivity in the spinal cord and motor cortex in amyotrophic lateral sclerosis. Arch. Neurol. 47, 1210–1216. Henkel, J.S., Engelhardt, J.I., Siklós, L., Simpson, E.P., Kim, S.H., Pan, T., Goodman, J.C., Siddique, T., Beers, D.R., Appel, S.H., 2004. Presence of dendritic cells, MCP-1, and activated microglia/macrophages in amyotrophic lateral sclerosis spinal cord tissue. Ann. Neurol. 55, 221–235. Kawamata, T., Akiyama, H., Yamada, T., McGeer, P.L., 1992. Immunologic reactions in amyotrophic lateral sclerosis brain and spinal cord tissue. Am. J. Pathol. 140, 691–707. Kim, J.I., Ho, I.C., Grusby, M.J., Glimcher, L.H., 1999. The transcription factor c-Maf controls the production of interleukin-4 but not other Th2 cytokines. Immunity 10, 745–751. Kira, J., Yamasaki, K., Kawano, Y., Kobayashi, T., 1997. Acute myelitis associated with hyperIgEemia and atopic dermatitis. J. Neurol. Sci. 148, 199–203. Kira, J., Osoegawa, M., Horiuchi, I., Murai, H., Minohara, M., Ohyagi, Y., Furuya, H., Tobimatsu, S., Yamasaki, K., Ochi, H., 2002. History of allergic disorders in common neurologic diseases in Japanese patients. Acta Neurol. Scand. 105, 215–220. Ochi, H., Osoegawa, M., Wu, X.M., Minohara, M., Horiuchi, I., Murai, H., Furuya, H., Kira, J., 2002. Increased IL-13 but not IL-5 production by CD4-positive T cells and CD8-positive T cells in multiple sclerosis during relapse phase. J. Neurol. Sci. 201, 45–51. Offen, D., Halevi, S., Orion, D., Mosberg, R., Stern-Goldberg, H., Melamed, E., Atlas, D., 1998. Antibodies from ALS patients inhibit dopamine release mediated by L-type calcium channels. Neurology 51, 1100–1103. Poloni, M., Facchetti, D., Mai, R., Micheli, A., Agnoletti, L., Francolini, G., Mora, G., Camana, C., Mazzini, L., Bachetti, T., 2000. Circulating levels of tumour necrosis factor-α and its soluble receptors are increased in the blood of patients with amyotrophic lateral sclerosis. Neurosci. Lett. 287, 211–214. Sekizawa, T., Openshaw, H., Ohbo, K., Sugamura, K., Itoyama, Y., Niland, J.C., 1998. Cerebrospinal fluid interleukin 6 in amyotrophic lateral sclerosis: Immunological parameter and comparison with inflammatory and noninflammatory central nervous system diseasesq. J. Neurol. Sci. 154, 194–199. Szczepanik, A.M., Funes, S., Petko, W., Ringheim, G.E., 2001. IL-4, IL-10 and IL-13 modulate A beta(1-42)-induced cytokine and chemokine production in primary murine microglia and a human monocyte cell line. J. Neuroimmunol. 113, 49–62. Tanaka, M., Kikuchi, H., Ishizu, T., Minohara, M., Osoegawa, M., Motomura, K., Tateishi, T., Ohyagi, Y., Kira, J., 2006. Intrathecal upregulation of G-CSF and its neuroprotective actions on motor neurons in amyotrophic lateral sclerosis. J. Neuropathol. Exp. Neurol. 65, 816–825. Yap, H.K., Cheung, W., Murugasu, B., Sim, S.K., Seah, C.C., Jordan, S.C., 1999. Th1 and Th2 cytokine mRNA profiles in childhood nephrotic syndrome: Evidence for increased IL-13 mRNA expression in relapse. J. Am. Soc. Nephrol. 10, 529–537. Zhu, J., Min, B., Hu-Li, J., Watson, C.J., Grinberg, A., Wang, Q., Killeen, N., Urban Jr., J.F., Guo, L., Paul, W.E., 2004. Conditional deletion of Gata3 shows its essential function in T(H)1-T(H)2 responses. Nat. Immunol. 5, 1157–1165.
Short communication
Increased IL-13-producing T cells in ALS: Positive correlations with disease severity and progression rate Nan Shi 1 , Yuji Kawano 1 , Takahisa Tateishi, Hitoshi Kikuchi, Manabu Osoegawa, Yasumasa Ohyagi, Jun-ichi Kira ⁎ Department of Neurology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan Received 22 June 2006; received in revised form 23 September 2006; accepted 2 October 2006
Abstract We measured the intracellular productions of IFNγ, IL-2, IL-4, IL-13 and TNFα in peripheral blood CD4+ and CD8+ T cells from 21 amyotrophic lateral sclerosis (ALS) patients, 14 disease controls (DC) with spinocerebellar degeneration and 16 healthy controls (HC). Only the percentages of CD4+IL-13+ and CD8+IL-13+ T cells were significantly higher in ALS patients than in DC and HC. The CD4+IL-13+ T cell percentages showed a significant negative correlation with the revised ALS functional rating scale scores and significant positive correlation with the disease progression rate, suggesting that IL-13 contributes to ALS. © 2006 Elsevier B.V. All rights reserved. Keywords: Amyotrophic lateral sclerosis; Interleukin-13; Neuroinflammation; CD4+ T cell; CD8+ T cell
1. Introduction
2. Subjects and methods
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that primarily affects the upper and lower motor neurons. Although its etiology remains to be elucidated, increasing evidence suggests that inflammatory processes may contribute to motor neuron damage in ALS. Specifically, proinflammatory cytokines, such as macrophage chemoattractant protein (MCP)-1 (Henkel et al., 2004; Tanaka et al., 2006), interleukin (IL)-5 (Tanaka et al., 2006), IL-6 (Sekizawa et al., 1998) and tumor necrosis factor-α (TNF-α) (Poloni et al., 2000), are increased in cerebrospinal fluid (CSF) and sera. Moreover, ALS spinal cord shows activation of microglia together with upregulation of MCP-1 mRNA (Henkel et al., 2004). These findings prompted us to study the intracellular cytokine productions of T helper type 1 (Th1) and Th2 cytokines in ALS patients.
2.1. Subjects
⁎ Corresponding author. Tel.: +81 92 642 5340; fax: +81 92 642 5352. E-mail address: [email protected] (J. Kira). 1 These authors contributed equally to this work. 0165-5728/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2006.10.001
A total of 21 Japanese patients (11 men, 10 women; mean age ± SD: 55 ± 15 years) with clinically definite ALS based on the El Escorial criteria (Brooks, 1994) who were not taking any drugs, 14 disease control patients with spinocerebellar degeneration (9 men, 5 women; mean age ± S.D.: 60 ± 14 years) and 16 healthy controls (12 men, 4 women; mean age ± S.D.: 44 ± 9 years) were enrolled in this study. At the time of blood sampling, none of the subjects were experiencing acute infections or inflammatory diseases. The disease duration of ALS was 24.8 ± 17.5 months. For the serum IgE assay, blood samples collected from the same 21 Japanese ALS patients as those enrolled for the intracellular cytokine assay and 59 healthy controls (30 men, 29 women; mean age ± S.D.: 47 ± 6 years) were evaluated. We evaluated the disabilities of the patients using the Revised ALS Functional Rating Scale (ALSFRS-R) (Cedarbaum et al., 1999), and found that their ALSFRS-R score was 34.6± 11.21 (mean ± S.D.; range: 0-48). We calculated the disease progression rate (DPR) as follows: DPR = (48− ALSFRS-
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R) / disease duration (months) (Tanaka et al., 2006). The DPR of the ALS patients was 0.82± 0.71 (mean± S.D.; range: 0.03–2.30). 2.2. Flow cytometry for intracellular cytokines The intracellular cytokine productions were studied by flow cytometry as described previously (Ochi et al., 2002). Briefly, peripheral blood mononuclear cells were collected from the subjects and stimulated with 25 ng/ml phorbol 12myristate 13-acetate (Sigma, St. Louis, MO) and 1 μg/ml of ionomycin (Sigma) in the presence of 10 μg/ml brefeldin A (Sigma) for 4 h at 37.5 °C. After washing with phosphatebuffered saline containing 0.1% bovine serum albumin (0.1% BSA-PBS), antibodies against cell surface markers were added for 15 min at room temperature in the dark. The cells were washed twice, permeabilized with FACS permeabilizing solution (Becton Dickinson, San Jose, CA) and incubated with antibodies against human cytokines or isotype-matched controls for 30 min. Finally, the cells were washed with 0.1% BSA-PBS and analyzed using an Epics XL System II (Coulter, Hialeah, FL). The monoclonal antibodies used were as follows: PC5-conjugated anti-CD4 (13B8.2; Becton Dickinson, San Jose, CA), PC5-conjugated anti-CD8 (B9.11; Becton Dickinson), FITC-conjugated anti-CD8 (B9.11; Beckman Coulter, Miami, FL), FITC-conjugated anti-IFNγ (25723.11; Becton Dickinson), PE-conjugated anti-IL-4 (3010.211; Becton Dickinson), PE-conjugated anti-TNF-α (6401.1111; Becton Dickinson), FITC-conjugated anti-IL-2 (5344.111; Becton Dickinson) and PE-conjugated anti-IL-13 (JES10-5A2; PharMingen, San Diego, CA). The percentages of cytokine-positive CD4+ and CD8+ cells were determined as the % cytokine-positive CD4+ population/total CD4+ population and % cytokine-positive CD8+ population/total CD8+ population, respectively.
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2.4. Statistical analysis Statistical analyses for comparisons of cell percentages were initially performed using the Kruskal–Wallis H test. When statistical significance was found, the Mann–Whitney U test was used to determine the statistical differences among the groups. Uncorrelated P values (Puncorr) were corrected by multiplying them by the number of comparisons (Bonferroni–Dunn's correction) to calculate corrected P values ( Pcorr). A Pcorr value of less than 0.05 was considered significant. Statistical analyses for comparisons of serum IgE concentrations were performed using the Mann–Whitney U test. The ALSFRS-R scores and IL-13-positive cell percentages among the CD4+ T cells showed normal distributions. However, the DPR and IL-13-positive cell percentages among the CD8+ T cells were not normally distributed. Therefore, the correlation between the cytokine-positive cell percentages in CD4+ T cells and ALSFRS-R scores was analyzed by Pearson's correlation test, while other correlations were analyzed by Spearman's rank correlation test. 3. Results 3.1. Intracellular cytokines in CD4+ T cells Among the cytokines examined, only the percentage of CD4+IL-13+ T cells was significantly higher in ALS patients than in healthy controls (Pcorr = 0.039) and disease controls (Pcorr = 0.024) (Table 1). The other percentages, including CD4+IL-4−IFNγ+, CD4+IL-4+IFNγ−, CD4+IL-2−TNFα+, CD4+IL-2+TNFα− and CD4+IL-2+TNFα+T cells, and the ratio of CD4+IL-4−IFNγ+T cells to CD4+IL-4+IFNγ−T cells (intracellular IFNγ/IL-4 ratio) did not differ significantly among the ALS patients, healthy controls and disease controls. 3.2. Intracellular cytokines in CD8+ T cells
2.3. Determination of total serum IgE Serum total IgE was measured using an enzyme-linked immunosorbent assay (ELISA) as described previously (Kira et al., 1997).
Similarly, only the percentage of CD8+IL-13+ T cells was significantly higher in ALS patients than in healthy controls (Pcorr = 0.006) (Table 1). The percentage of CD8+IL-13+ T cells was also higher in ALS patients than in disease controls,
Table 1 Intracellular cytokine productions in peripheral blood CD4+ and CD8+ T cells IL-4-IFN-γ+
IL-4+IFN-γ-
IL-4+IFN-γ+
CD4+T cells HC (n = 16) DC (n = 14) ALS (n = 21)
22.13 ± 7.16 25.59 ± 8.95 20.72 ± 6.23
1.24 ± 0.46 1.02 ± 0.6 1.46 ± 0.88
0.55 ± 0.46 0.22 ± 0.23 0.30 ± 0.24
CD8+T cells HC (n = 16) DC (n = 14) ALS (n = 21)
55.91 ± 16.48 64.24 ± 18.12 57.59 ± 17.75
0.91 ± 0.66 1.65 ± 2.24 1.82 ± 2.56
0.33 ± 0.33 0.34 ± 0.20 0.39 ± 0.37
IFN-γ+/IL-4+
IL-13+
IL-2-TNF-α+
IL-2+TNF-α-
IL-2+TNF-α+
20.16 ± 13.82 40.17 ± 53.55 23.76 ± 26.29
4.21 ± 2.58 3.54 ± 1.73 6.96 ± 3.97 a
20.38 ± 6.30 22.24 ± 9.99 17.97 ± 5.20
9.44 ± 3.56 12.74 ± 6.7 9.54 ± 3.85
31.03 ± 11.47 33.82 ± 8.21 32.80 ± 11.88
176.88 ± 293.80 275.0 ± 489.15 124.32 ± 143.93
2.85 ± 1.66 4.64 ± 6.19 6.94 ± 5.67 b
32.67 ± 11.47 36.89 ± 14.66 37.26 ± 17.95
7.44 ± 4.76 7.31 ± 4.70 5.05 ± 3.91
16.68 ± 9.36 22.67 ± 9.78 18.02 ± 11.46
ALS, amyotrophic lateral sclerosis patients; HC, healthy controls; DC, disease controls. a Significant difference vs. HC ( Pcorr b 0.05) or DC ( Pcorr b 0.05). b Significant difference vs. HC ( Pcorr b 0.05).
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Fig. 1. Correlations between the peripheral blood IL-13+ cell percentages and the Revised ALS Functional Rating Scale (ALSFRS-R) scores (A and B) and between the peripheral blood IL-13+ cell percentages and the disease progression rate (C and D).
but the difference did not reach statistical significance (Pcorr N 0.1). 3.3. Total serum IgE concentration The mean serum IgE concentration was higher in ALS patients (mean ± S.D.: 181 ± 242 IU/ml) than in healthy controls (mean ± S.D.: 117 ± 148 IU/ml), but the difference did not reach statistical significance (P N 0.05) due to the small sample numbers. 3.4. Correlations between the ALSFRS-R score and IL-13 production, and between the DPR and IL-13 production The IL-13-positive cell percentages showed a significant negative correlation with the ALSFRS-R scores in CD4+ T cells (r = − 0.497, p = 0.022), but not in CD8+ T cells (Fig. 1). In addition, the IL-13-positive cell percentages showed a significant positive correlation with the DPR in CD4+ T cells (r = 0.496, p = 0.027), but not in CD8+ T cells (Fig. 1). 4. Discussion In the present study, we have revealed for the first time that IL-13 production is upregulated in peripheral blood CD4+ and CD8+ T cells in ALS and that the number of IL13-producing CD4+ T cells is significantly associated with the disease severity and progression rate.
Although no statistically significant difference in the serum IgE concentrations was observed between ALS patients and healthy controls due to the small sample numbers, the mean serum IgE concentration was higher in ALS patients than in healthy controls. The percentage of CD4+IL13+ T cells in ALS patients was as high as that seen in multiple sclerosis patients at relapse, in which we previously reported its upregulation (Ochi et al., 2002). Moreover, IL-13 production was positively correlated with the DPR. These findings suggest that upregulation of IL-13 production in ALS patients has some biological relevance and that IL-13 could be involved in the neurodegeneration observed in ALS. We detected no differences in other cytokine productions. These findings suggest that systemic immune activation, especially Th1 type immune activation, is not involved in ALS. Among the Th2 cytokines examined, IL-13 was upregulated in the present study, whereas IL-4 was not. Accumulating evidence has indicated that each Th2 cytokine has its own distinct regulation system. For example, c-Maf is essential for the expression of IL-4, but not IL-13 (Kim et al., 1999). On the contrary, IL-13 is regulated by GATA-3, while IL-4 is not (Zhu et al., 2004). Therefore, the isolated IL-13 upregulation seen in ALS is probably based on differences among the regulation systems of Th2 cytokines. Such isolated IL-13 upregulation was also observed in nephrotic syndrome at the relapse phase (Yap et al., 1999), where expression of IL-13, but not IL-2, IL-4 or INF-γ, was
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increased in T cells and considered to contribute to disease exacerbation through immunomodulatory effects on macrophages and B cells. IL-13 drives humoral immunity and potentiates IgE synthesis. Humoral immunity is thought to be involved in ALS, since IgG is deposited in the spinal cord and motor cortex in ALS (Engelhardt and Appel, 1990) and autoantibodies to calcium channels are present (Offen et al., 1998). In addition, the incidence of history of past and concomitant asthma is significantly increased in patients with motor neuron diseases in Japan, as evaluated in a largescale prospective study (Kira et al., 2002). Thus, the increased number of IL-13-producing T cells in ALS may be related to such abnormalities in humoral immunity. On the other hand, IL-13 enhances MCP-1 expression in monocytes/macrophages (Szczepanik et al., 2001). MCP-1 attracts monocytes/macrophages and CCR2-bearing Th2 cells and activates monocytes and microglia, thereby leading to the production of proinflammatory cytokines and nitric oxide. Furthermore, MCP-1 in CSF is positively correlated with disease severity (Tanaka et al., 2006). Therefore, IL-13 may aggravate ALS through MCP-1 induction and subsequent glial inflammation. CD4+ and CD8+ T cells are known to be present in the ALS spinal cord (Kawamata et al., 1992). IL-13 upregulates the expression of vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells, which plays a crucial role in mediating the adhesion and migration of inflammatory cells in the central nervous system (CNS). Moreover, IL-13 enhances the expression of MHC class II molecules in monocytes/macrophages (Cash et al., 1994), thereby potentiating the capacity of these cells to present relevant antigens. IL-13 may therefore facilitate the CNS inflammatory cascade in ALS. Our present results suggest that IL-13 contributes to the aggravation of ALS, probably through potentiation of inflammatory components of the disease. Acknowledgements This work was supported in part by grants from the Ministry of Education, Culture, Science and Technology of Japan, the Neuroimmunological Disease Research Committee, the Ministry of Health, Labor and Welfare of Japan and the Japan ALS Association. References Brooks, B.R., 1994. El Escorial World Federation of Neurology criteria for the diagnosis of amyotrophic lateral screrosis. Subcommittee on Motor Neuron Diseases/Amyotrophic Lateral Sclerosis of the World Federation of Neurology Research Group on Neuromuscular Diseases and the El Escorial “Clinical limits of amyotrophic lateral screlosis” workshop contributors. J. Neurol. Sci. 124, 96–107 (Suppl).
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