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Increased serum levels of soluble CD14 indicate stable multiple sclerosis
Journal of Neuroimmunology 181 (2006) 145 – 149 www.elsevier.com/locate/jneuroim
Clinical neuroimmunology
Increased serum levels of soluble CD14 indicate stable multiple sclerosis Andreas Lutterotti, Bettina Kuenz, Viktoria Gredler, Michael Khalil, Rainer Ehling, Claudia Gneiss, Robert Egg, Florian Deisenhammer, Thomas Berger, Markus Reindl ⁎ Clinical Department of Neurology, Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, Austria Received 2 June 2006; received in revised form 5 September 2006; accepted 5 September 2006
Abstract The aim of this study was to evaluate a possible role of soluble CD14 (sCD14) in multiple sclerosis (MS). We found that sCD14 serum levels measured by ELISA were higher in MS patients compared to neurological and healthy controls. Within the MS group sCD14 levels were increased in relapsing–remitting and secondary progressive MS compared to primary progressive MS. Furthermore, sCD14 concentrations were increased during stable disease. An increased expression of sCD14 was also detected after treatment with interferon-beta. In summary, we report evidence that serum sCD14 levels are increased in MS and correlate inversely with disease activity in relapsing MS patients. © 2006 Elsevier B.V. All rights reserved. Keywords: Multiple sclerosis; Soluble CD14; Disease activity; Innate immunity
1. Introduction Multiple sclerosis (MS), a chronic inflammatory demyelinating disease of the central nervous system (CNS), is generally assumed to result from an autoimmune attack directed against myelin components (Sospedra and Martin, 2005). There is increasing evidence that, besides adaptive immunity, the innate immune system plays a substantial role in the induction of autoimmune diseases (Kerfoot et al., 2004; Pasare and Medzhitov, 2003; Waldner et al., 2004). Recently, the innate immune receptor CD14 was shown to be up-regulated in the brain of MS patients and CD14 expression correlated with disease activity in EAE (Walter et al., 2006; Zekki et al., 2002). CD14 is a membrane glycophosphatidylinositol-anchored receptor (mCD14) mainly expressed on monocytes, macrophages and neutrophil granulocytes (Pugin et al., 1994; Wright et al., 1990). As a pattern recognition receptor of microbial products CD14 is known to be an essential mediator of inflammation in innate host defense (Pugin et al., 1994). CD14 has also been shown to be involved in the non-inflammatory
phagocytosis of apoptotic cells by macrophages (Devitt et al., 1998; Schlegel et al., 1999). A soluble form of CD14 (sCD14), which stems from protease mediated shedding from cell membranes but also from secretion by hepatocytes, can be detected in serum or plasma (Bas et al., 2004; Bazil et al., 1986; Pan et al., 2000). Expression of mCD14 and release of sCD14 are regulated by bacterial infections and cytokines (Landmann et al., 2000). Elevated serum levels of sCD14 have been reported in MS patients but also in other organ specific autoimmune diseases (Bas et al., 2004; Brettschneider et al., 2002; Egerer et al., 2000; Nockher et al., 1994; Takeshita et al., 2000; Wuthrich et al., 1992; Yu et al., 1998). In this study we were interested whether serum levels of sCD14 correlate with the clinical course, disease activity and severity of MS. Furthermore, we were interested whether immunomodulatory therapies, such as interferon-beta (IFNβ), influence sCD14 levels. 2. Patients and methods 2.1. MS patients and controls
⁎ Corresponding author. Tel.: +43 512/504 24363; fax: +43 512/504 24266. E-mail address: [email protected] (M. Reindl). 0165-5728/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2006.09.002
Serum levels of sCD14 were analyzed in 165 patients with clinically definite MS (McDonald et al., 2001)
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Table 1 Demographic and clinical data of MS patients and controls Patient group
RRMS
SPMS
PPMS
OND
HC
p-value
Number of patients Females Age1,2 Disease duration1,2 EDSS1,3 Progression index1,4 MSSS5 Number of relapses1 Relapse rate1,6 No treatment IMD7 treatment IS8 treatment Acute relapse Stable disease Progression
96 62 (65%) 36 (19–70) 8 (1–35) 2 (0–6.0) 0.2 (0–1.8) 2.0 (0.1–8.9) 4 (1–31) 0.6 (0.1–2) 47 (49%) 45 (47%) 4 (4%) 21 75 0
50 30 (60%) 49 (24–67) 19 (5–35) 6 (2–8.5) 0.3 (0.1–1.3) 5.9 (0.9–9.6) 6 (1–29) 0.4 (0.1–2) 25 (50%) 21 (42%) 4 (8%) 6 3 41
19 9 (47%) 58 (32–69) 13 (2–22) 4 (2–6.5) 0.4 (0.2–1.3) 6.3 (2.9–9.3) 0 0 4 (21%) 10 (53%) 5 (26%) 0 0 19
22 12 (55%) 35 (17–79)
50 33 (66%) 40 (21–68)
ns ns
RRMS = relapsing–remitting MS; SPMS = secondary progressive MS; PPMS = primary progressive MS; OND = other neurological diseases; HC = healthy controls. 1 Median (range); 2years; 3EDSS = expanded disability status scale (Kurtzke, 1983); 4progression index = EDSS/disease duration; 5Multiple sclerosis severity score (Roxburgh et al., 2005); 6relapse rate = number of relapses/disease duration; 7immunomodulatory treatment; 8immunosuppressive treatment.
(Table 1). In all patients serum samples were drawn before corticosteroid treatment. To control for systemic infections we analyzed whole blood leukocyte counts and blood levels of acute C-reactive protein (CRP). As control groups we analyzed sCD14 serum levels in 22 patients with other neurological diseases (OND; 3 bacterial meningitis, 5 viral meningitis, 2 viral encephalitis, 1 myelitis, 11 headache) and 50 healthy controls (Table 1). To elucidate a possible influence of immunomodulatory treatments on sCD14 serum levels we further analyzed an independent cohort of 50 MS patients, where serum samples were prospectively collected before treatment was started and after one year (IFNβ 29, Glatiramer-acetate 8 and intravenous immunoglobulins (IVIG) 2 patients) and in 11 patients who received no therapy between the two time points (Table 2).
2.2. Collection of samples and sample preparation Whole blood was collected with informed consent by peripheral vein puncture (Sarstedt Nuembrecht, Germany). Serum samples were stored at −20 °C until analysis was performed. EDTA and heparinized blood were used to analyze complete blood count and CRP levels. 2.3. Determination of sCD14 levels by ELISA The serum levels of sCD14 were determined using a commercially available enzyme-linked immunosorbent assay (ELISA) (IBL, Hamburg, Germany). Serum samples
Table 2 Demographic and clinical data of MS patients prospectively analyzed for the influence of immunomodulatory therapies on serum sCD14 levels Patient group
IFNβ
Other IM
No IM
p-value
Number of patients Females Age1,2 Disease duration1,2 EDSS1,3 Progression index1,4 MSSS1,5 Number of relapses1 Relapse rate1,6
29 19 (66%) 35 (19–44) 3.5 (0.5–21) 1.5 (0–3.5) 0.3 (0–1.6) 2.7 (0.2–7.1) 3 (2–10) 1.0 (0.3–4.4)
10 4 (40%) 29 (22–38) 4.6 (1–16) 1.5 (1–3) 0.2 (0.1–0.6) 1.8 (0.8–6.2) 4.5 (2–15) 1.0 (0.4–2.1)
11 8 (73%) 32 (25–54) 4.9 (1–13) 1 (0–1.5) 0.2 (0–1.5) 1.8 (0.2–4.7) 3 (2–6) 0.8 (0.3–2.1)
ns ns ns ns ns ns ns ns
IFNβ = interferon-beta; other IM = other immunomodulatory treatments (Glatiramer-acetate n = 8 and intravenous immunoglobulins n = 2); no IM = no immunomodulatory treatments. 1 Median (range); 2years; 3EDSS = expanded disability status scale (Kurtzke, 1983); 4progression index = EDSS/disease duration; Multiple sclerosis severity score (Roxburgh et al., 2005); 6relapse rate = number of relapses/ disease duration.
Fig. 1. Serum levels of sCD14 in MS subgroups (RRMS, SPMS and PPMS), patients with other neurological disease (OND) and healthy controls (HC). Data are shown as individual replicates and as box-plot. Medians are indicated by horizontal bars within the boxes.
A. Lutterotti et al. / Journal of Neuroimmunology 181 (2006) 145–149
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were diluted 1:201 and analyzed in accordance with the instructions of the manufacturer. All serial samples from each individual taken at different time points were analyzed on the same ELISA plate. Each sample was measured in duplicate. 2.4. Statistical analysis Statistical analysis was performed using SPSS (release 11.0, SPSS Inc., USA) and PRISM 4 (GraphPad Software Inc, USA) software. Distribution of groups was analyzed by Kolmogorov–Smirnov test. Between-group comparisons of not normally distributed data were made using Kruskal– Wallis Test with Dunn's multiple comparison test. Normally distributed data were analyzed by a univariate general model using sex, treatment, age, disease duration, EDSS and relapse rate as covariates. Correlation of parameters was performed using Spearman non-parametric correlation. 3. Results 3.1. Increased serum sCD14 levels in MS patients Serum levels of sCD14 were increased in RRMS, SPMS and PPMS compared to healthy controls and in RRMS and SPMS compared to OND (Fig. 1). Within the MS group sCD14 levels were higher in RRMS and SPMS than in PPMS. Next we correlated sCD14 levels with clinical variables of disease activity in MS patients. As shown in Fig. 2 we found the highest serum sCD14 levels in patients with stable disease compared to patients at the time of an acute relapse or in
Fig. 2. Serum levels of sCD14 in MS patients with acute relapse, stable disease and progression. Data are shown as individual replicates and as boxplot. Medians are indicated by horizontal bars within the boxes.
Fig. 3. Influence of relapses on serum levels of sCD14. Blood samples of MS patients were taken 3 months before an acute relapse (baseline) and at the time of relapse. Data are shown as box-plot. Medians are indicated by horizontal bars within the boxes.
progression. Furthermore, serum sCD14 levels in patients at the time of an acute relapse were lower than in progression. Statistical analysis revealed that these differences in sCD14 levels were not influenced by age, sex, disease duration, EDSS, progression index or MSSS, relapses and MS therapies. The influence of relapses on sCD14 levels was further analyzed in 10 MS patients of whom additional samples taken 3 months before relapse (baseline) were available. In this small group of patients we observed a significant decrease of sCD14 levels from baseline to relapse (Fig. 3). None of the patients in relapse had any clinical or laboratory sign of a current infection. In the whole MS population only 5 (3%) patients had a slightly elevated leukocyte count, without clinical signs of infection. sCD14 levels correlated positively with CRP (r = 0.45, p = 0.003), the percentage of neutrophils (r = 0.27, p = 0.002) and negatively with the percentage of
Fig. 4. Influence of MS therapies on serum levels of sCD14. Blood samples of MS patients treated with IFNβ, other immunomodulatory therapies and untreated MS patients were taken before immunomodulatory therapy was started and one year later. Data are shown as delta sCD14 (1-year baseline). Data are shown as box-plots and medians are indicated by horizontal bars within the boxes.
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lymphocytes (r = −0.27, p = 0.003). There was no correlation between sCD14 and the monocyte count. 3.2. Influence of immunomodulatory treatment on sCD14 serum levels Treatment with immunomodulatory or immunosuppressive drugs had no significant impact on sCD14 serum levels in the overall MS population. However, in a subgroup analysis we found that sCD14 levels were highest in patients with IFNβ treatment (p b 0.05, data not shown). We therefore analyzed the influence of IFNβ treatment on sCD14 serum levels in an independent prospective cohort of 50 MS patients. IFNβ treatment significantly increased sCD14 serum levels in MS patients after one year of treatment compared to baseline (p b 0.001), whereas there was no significant increase of sCD14 levels in patients receiving Glatiramer-acetate, IVIG or patients without therapy (Fig. 4). The change in serum sCD14 levels did not correlate with changes in relapse rate, EDSS, progression index or MSSS. 4. Discussion In this study we report elevated serum levels of sCD14 in patients with MS compared to patients with OND and healthy blood donors. Consistent with our results elevated levels of sCD14 have been found in MS patients and in several autoimmune diseases (Bas et al., 2004; Brettschneider et al., 2002; Egerer et al., 2000; Nockher et al., 1994; Yu et al., 1998). The levels of sCD14 in our healthy controls were comparable to previously published data (Ayaslioglu et al., 2005; Brettschneider et al., 2002; Egerer et al., 2000). Our main finding is that sCD14 levels correlate inversely with the clinical activity of the disease in MS patients. sCD14 levels were significantly increased in MS patients with stable disease compared to patients with an acute relapse or a progressive disease. The difference in sCD14 levels between PPMS and bout onset MS patients might reflect the different inflammatory activity between the two disease courses. Corroborating this result, we observed a decrease in sCD14 levels during relapse in a small number of longitudinally analyzed patients. Thus, the differential expression of sCD14 in MS patients might reflect the current peripheral inflammatory activity of the disease rather than the clinical type of disease course. Brettschneider reported higher sCD14 levels in RRMS compared to PPMS patients without statistical significance between the two groups, but their sample size was considerably smaller (Brettschneider et al., 2002). In the same study they found no difference in sCD14 between patients in relapse and patients in remission. The differences in sCD14 levels observed in our study may also reflect a differential activation and role of the innate immune system in different inflammatory activities of MS. This differential activation of sCD14 might be a result of current infections. In MS patients systemic infections are an important risk factor for exacerbations (Buljevac et al., 2002;
Sibley et al., 1985). Although we did not have any clinical or laboratory evidence of a current infection at the time of relapse in our patients we cannot exclude sub-clinical infections preceding the time point where the blood sample was drawn. The fact that sCD14 levels are elevated in patients during stable disease suggests a protective role of the soluble protein in MS patients. In EAE, CD14 deficient mice have a more severe disease and exhibit a markedly enhanced infiltration of monocytes and neutrophils within the CNS (Walter et al., 2006). In vivo and in vitro studies support the hypothesis that sCD14 has systemic anti-inflammatory effects by reducing the response of monocytes to LPS (Haziot et al., 1994; Kitchens and Thompson, 2005; Kitchens et al., 2001). Furthermore sCD14 has the capacity to negatively regulate T lymphocyte activation and function through direct interaction with T cells and to modulate humoral immune response by interfering with CD40 signaling in B cells (Arias et al., 2000; Rey Nores et al., 1999). In the overall population the correlation of sCD14 with the clinical activity was independent of treatment effect as it was found even in patients without therapy. Brettschneider reported an induction of sCD14 by IFNβ treatment in a serial analysis of PPMS patients (Brettschneider et al., 2002). Therapy with IFNβ for one year induced higher sCD14 serum levels in our RRMS patients as well. It is not clear whether the rise in sCD14 serum levels is due to a direct effect of IFNβ or whether it is an indirect result of a more stable disease course. So far only few data exist on the effect of IFNβ on the expression of the CD14 gene or the soluble protein. A gene expression analysis of IFNβ treated monocytes reported a significant down-regulation of CD14 gene expression in vitro (Wandinger et al., 2001). Differing to this result other groups found that therapy with IFNβ induces a higher expression of CD14 on monocytes and increases the percentage of these CD14 positive cells (Mirowska et al., 2003; Then Bergh et al., 2004). In summary, we provide evidence that serum sCD14 levels and the activation of innate immune response might reflect changes in MS disease activity. References Arias, M.A., Rey Nores, J.E., Vita, N., Stelter, F., Borysiewicz, L.K., Ferrara, P., Labeta, M.O., 2000. Cutting edge: human B cell function is regulated by interaction with soluble CD14: opposite effects on IgG1 and IgE production. J. Immunol. 164, 3480–3486. Ayaslioglu, E., Tekeli, E., Birengel, S., 2005. Significant elevation of serum soluble cd14 levels in patients with brucellosis. Jpn. J. Infect. Dis. 58, 11–14. Bas, S., Gauthier, B.R., Spenato, U., Stingelin, S., Gabay, C., 2004. CD14 is an acute-phase protein. J. Immunol. 172, 4470–4479. Bazil, V., Horejsi, V., Baudys, M., Kristofova, H., Strominger, J.L., Kostka, W., Hilgert, I., 1986. Biochemical characterization of a soluble form of the 53kDa monocyte surface antigen. Eur. J. Immunol. 16, 1583–1589. Brettschneider, J., Ecker, D., Bitsch, A., Bahner, D., Bogumil, T., Dressel, A., Elitok, E., Kitze, B., Poser, S., Weber, F., Tumani, H., 2002. The macrophage activity marker sCD14 is increased in patients with multiple sclerosis and upregulated by interferon beta-1b. J. Neuroimmunol. 133, 193–197.
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Roxburgh, R.H., Seaman, S.R., Masterman, T., Hensiek, A.E., Sawcer, S.J., Vukusic, S., Achiti, I., Confavreux, C., Coustans, M., le Page, E., Edan, G., McDonnell, G.V., Hawkins, S., Trojano, M., Liguori, M., Cocco, E., Marrosu, M.G., Tesser, F., Leone, M.A., Weber, A., Zipp, F., Miterski, B., Epplen, J.T., Oturai, A., Sorensen, P.S., Celius, E.G., Lara, N.T., Montalban, X., Villoslada, P., Silva, A.M., Marta, M., Leite, I., Dubois, B., Rubio, J., Butzkueven, H., Kilpatrick, T., Mycko, M.P., Selmaj, K.W., Rio, M.E., Sa, M., Salemi, G., Savettieri, G., Hillert, J., Compston, D.A., 2005. Multiple Sclerosis Severity Score: using disability and disease duration to rate disease severity. Neurology 64, 1144–1151. Schlegel, R.A., Krahling, S., Callahan, M.K., Williamson, P., 1999. CD14 is a component of multiple recognition systems used by macrophages to phagocytose apoptotic lymphocytes. Cell Death Differ. 6, 583–592. Sibley, W.A., Bamford, C.R., Clark, K., 1985. Clinical viral infections and multiple sclerosis. Lancet 1, 1313–1315. Sospedra, M., Martin, R., 2005. Immunology of multiple sclerosis. Annu. Rev. Immunol. 23, 683–747. Takeshita, S., Nakatani, K., Tsujimoto, H., Kawamura, Y., Kawase, H., Sekine, I., 2000. Increased levels of circulating soluble CD14 in Kawasaki disease. Clin. Exp. Immunol. 119, 376–381. Then Bergh, F., Dayyani, F., Ziegler-Heitbrock, L., 2004. Impact of type-Iinterferon on monocyte subsets and their differentiation to dendritic cells. An in vivo and ex vivo study in multiple sclerosis patients treated with interferon-beta. J. Neuroimmunol. 146, 176–188. Waldner, H., Collins, M., Kuchroo, V.K., 2004. Activation of antigenpresenting cells by microbial products breaks self tolerance and induces autoimmune disease. J. Clin. Invest. 113, 990–997. Walter, S., Doering, A., Letiembre, M., Liu, Y., Hao, W., Diem, R., Bernreuther, C., Glatzel, M., Engelhardt, B., Fassbender, K., 2006. The LPS receptor, CD14 in experimental autoimmune encephalomyelitis and multiple sclerosis. Cell. Physiol. Biochem. 17, 167–172. Wandinger, K.P., Sturzebecher, C.S., Bielekova, B., Detore, G., Rosenwald, A., Staudt, L.M., McFarland, H.F., Martin, R., 2001. Complex immunomodulatory effects of interferon-beta in multiple sclerosis include the upregulation of T helper 1-associated marker genes. Ann. Neurol. 50, 349–357. Wright, S.D., Ramos, R.A., Tobias, P.S., Ulevitch, R.J., Mathison, J.C., 1990. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249, 1431–1433. Wuthrich, B., Kagi, M.K., Joller-Jemelka, H., 1992. Soluble CD14 but not interleukin-6 is a new marker for clinical activity in atopic dermatitis. Arch. Dermatol. Res. 284, 339–342. Yu, S., Nakashima, N., Xu, B.H., Matsuda, T., Izumihara, A., Sunahara, N., Nakamura, T., Tsukano, M., Matsuyama, T., 1998. Pathological significance of elevated soluble CD14 production in rheumatoid arthritis: in the presence of soluble CD14, lipopolysaccharides at low concentrations activate RA synovial fibroblasts. Rheumatol. Int. 17, 237–243. Zekki, H., Feinstein, D.L., Rivest, S., 2002. The clinical course of experimental autoimmune encephalomyelitis is associated with a profound and sustained transcriptional activation of the genes encoding toll-like receptor 2 and CD14 in the mouse CNS. Brain Pathol. 12, 308–319.
Clinical neuroimmunology
Increased serum levels of soluble CD14 indicate stable multiple sclerosis Andreas Lutterotti, Bettina Kuenz, Viktoria Gredler, Michael Khalil, Rainer Ehling, Claudia Gneiss, Robert Egg, Florian Deisenhammer, Thomas Berger, Markus Reindl ⁎ Clinical Department of Neurology, Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, Austria Received 2 June 2006; received in revised form 5 September 2006; accepted 5 September 2006
Abstract The aim of this study was to evaluate a possible role of soluble CD14 (sCD14) in multiple sclerosis (MS). We found that sCD14 serum levels measured by ELISA were higher in MS patients compared to neurological and healthy controls. Within the MS group sCD14 levels were increased in relapsing–remitting and secondary progressive MS compared to primary progressive MS. Furthermore, sCD14 concentrations were increased during stable disease. An increased expression of sCD14 was also detected after treatment with interferon-beta. In summary, we report evidence that serum sCD14 levels are increased in MS and correlate inversely with disease activity in relapsing MS patients. © 2006 Elsevier B.V. All rights reserved. Keywords: Multiple sclerosis; Soluble CD14; Disease activity; Innate immunity
1. Introduction Multiple sclerosis (MS), a chronic inflammatory demyelinating disease of the central nervous system (CNS), is generally assumed to result from an autoimmune attack directed against myelin components (Sospedra and Martin, 2005). There is increasing evidence that, besides adaptive immunity, the innate immune system plays a substantial role in the induction of autoimmune diseases (Kerfoot et al., 2004; Pasare and Medzhitov, 2003; Waldner et al., 2004). Recently, the innate immune receptor CD14 was shown to be up-regulated in the brain of MS patients and CD14 expression correlated with disease activity in EAE (Walter et al., 2006; Zekki et al., 2002). CD14 is a membrane glycophosphatidylinositol-anchored receptor (mCD14) mainly expressed on monocytes, macrophages and neutrophil granulocytes (Pugin et al., 1994; Wright et al., 1990). As a pattern recognition receptor of microbial products CD14 is known to be an essential mediator of inflammation in innate host defense (Pugin et al., 1994). CD14 has also been shown to be involved in the non-inflammatory
phagocytosis of apoptotic cells by macrophages (Devitt et al., 1998; Schlegel et al., 1999). A soluble form of CD14 (sCD14), which stems from protease mediated shedding from cell membranes but also from secretion by hepatocytes, can be detected in serum or plasma (Bas et al., 2004; Bazil et al., 1986; Pan et al., 2000). Expression of mCD14 and release of sCD14 are regulated by bacterial infections and cytokines (Landmann et al., 2000). Elevated serum levels of sCD14 have been reported in MS patients but also in other organ specific autoimmune diseases (Bas et al., 2004; Brettschneider et al., 2002; Egerer et al., 2000; Nockher et al., 1994; Takeshita et al., 2000; Wuthrich et al., 1992; Yu et al., 1998). In this study we were interested whether serum levels of sCD14 correlate with the clinical course, disease activity and severity of MS. Furthermore, we were interested whether immunomodulatory therapies, such as interferon-beta (IFNβ), influence sCD14 levels. 2. Patients and methods 2.1. MS patients and controls
⁎ Corresponding author. Tel.: +43 512/504 24363; fax: +43 512/504 24266. E-mail address: [email protected] (M. Reindl). 0165-5728/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2006.09.002
Serum levels of sCD14 were analyzed in 165 patients with clinically definite MS (McDonald et al., 2001)
146
A. Lutterotti et al. / Journal of Neuroimmunology 181 (2006) 145–149
Table 1 Demographic and clinical data of MS patients and controls Patient group
RRMS
SPMS
PPMS
OND
HC
p-value
Number of patients Females Age1,2 Disease duration1,2 EDSS1,3 Progression index1,4 MSSS5 Number of relapses1 Relapse rate1,6 No treatment IMD7 treatment IS8 treatment Acute relapse Stable disease Progression
96 62 (65%) 36 (19–70) 8 (1–35) 2 (0–6.0) 0.2 (0–1.8) 2.0 (0.1–8.9) 4 (1–31) 0.6 (0.1–2) 47 (49%) 45 (47%) 4 (4%) 21 75 0
50 30 (60%) 49 (24–67) 19 (5–35) 6 (2–8.5) 0.3 (0.1–1.3) 5.9 (0.9–9.6) 6 (1–29) 0.4 (0.1–2) 25 (50%) 21 (42%) 4 (8%) 6 3 41
19 9 (47%) 58 (32–69) 13 (2–22) 4 (2–6.5) 0.4 (0.2–1.3) 6.3 (2.9–9.3) 0 0 4 (21%) 10 (53%) 5 (26%) 0 0 19
22 12 (55%) 35 (17–79)
50 33 (66%) 40 (21–68)
ns ns
RRMS = relapsing–remitting MS; SPMS = secondary progressive MS; PPMS = primary progressive MS; OND = other neurological diseases; HC = healthy controls. 1 Median (range); 2years; 3EDSS = expanded disability status scale (Kurtzke, 1983); 4progression index = EDSS/disease duration; 5Multiple sclerosis severity score (Roxburgh et al., 2005); 6relapse rate = number of relapses/disease duration; 7immunomodulatory treatment; 8immunosuppressive treatment.
(Table 1). In all patients serum samples were drawn before corticosteroid treatment. To control for systemic infections we analyzed whole blood leukocyte counts and blood levels of acute C-reactive protein (CRP). As control groups we analyzed sCD14 serum levels in 22 patients with other neurological diseases (OND; 3 bacterial meningitis, 5 viral meningitis, 2 viral encephalitis, 1 myelitis, 11 headache) and 50 healthy controls (Table 1). To elucidate a possible influence of immunomodulatory treatments on sCD14 serum levels we further analyzed an independent cohort of 50 MS patients, where serum samples were prospectively collected before treatment was started and after one year (IFNβ 29, Glatiramer-acetate 8 and intravenous immunoglobulins (IVIG) 2 patients) and in 11 patients who received no therapy between the two time points (Table 2).
2.2. Collection of samples and sample preparation Whole blood was collected with informed consent by peripheral vein puncture (Sarstedt Nuembrecht, Germany). Serum samples were stored at −20 °C until analysis was performed. EDTA and heparinized blood were used to analyze complete blood count and CRP levels. 2.3. Determination of sCD14 levels by ELISA The serum levels of sCD14 were determined using a commercially available enzyme-linked immunosorbent assay (ELISA) (IBL, Hamburg, Germany). Serum samples
Table 2 Demographic and clinical data of MS patients prospectively analyzed for the influence of immunomodulatory therapies on serum sCD14 levels Patient group
IFNβ
Other IM
No IM
p-value
Number of patients Females Age1,2 Disease duration1,2 EDSS1,3 Progression index1,4 MSSS1,5 Number of relapses1 Relapse rate1,6
29 19 (66%) 35 (19–44) 3.5 (0.5–21) 1.5 (0–3.5) 0.3 (0–1.6) 2.7 (0.2–7.1) 3 (2–10) 1.0 (0.3–4.4)
10 4 (40%) 29 (22–38) 4.6 (1–16) 1.5 (1–3) 0.2 (0.1–0.6) 1.8 (0.8–6.2) 4.5 (2–15) 1.0 (0.4–2.1)
11 8 (73%) 32 (25–54) 4.9 (1–13) 1 (0–1.5) 0.2 (0–1.5) 1.8 (0.2–4.7) 3 (2–6) 0.8 (0.3–2.1)
ns ns ns ns ns ns ns ns
IFNβ = interferon-beta; other IM = other immunomodulatory treatments (Glatiramer-acetate n = 8 and intravenous immunoglobulins n = 2); no IM = no immunomodulatory treatments. 1 Median (range); 2years; 3EDSS = expanded disability status scale (Kurtzke, 1983); 4progression index = EDSS/disease duration; Multiple sclerosis severity score (Roxburgh et al., 2005); 6relapse rate = number of relapses/ disease duration.
Fig. 1. Serum levels of sCD14 in MS subgroups (RRMS, SPMS and PPMS), patients with other neurological disease (OND) and healthy controls (HC). Data are shown as individual replicates and as box-plot. Medians are indicated by horizontal bars within the boxes.
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were diluted 1:201 and analyzed in accordance with the instructions of the manufacturer. All serial samples from each individual taken at different time points were analyzed on the same ELISA plate. Each sample was measured in duplicate. 2.4. Statistical analysis Statistical analysis was performed using SPSS (release 11.0, SPSS Inc., USA) and PRISM 4 (GraphPad Software Inc, USA) software. Distribution of groups was analyzed by Kolmogorov–Smirnov test. Between-group comparisons of not normally distributed data were made using Kruskal– Wallis Test with Dunn's multiple comparison test. Normally distributed data were analyzed by a univariate general model using sex, treatment, age, disease duration, EDSS and relapse rate as covariates. Correlation of parameters was performed using Spearman non-parametric correlation. 3. Results 3.1. Increased serum sCD14 levels in MS patients Serum levels of sCD14 were increased in RRMS, SPMS and PPMS compared to healthy controls and in RRMS and SPMS compared to OND (Fig. 1). Within the MS group sCD14 levels were higher in RRMS and SPMS than in PPMS. Next we correlated sCD14 levels with clinical variables of disease activity in MS patients. As shown in Fig. 2 we found the highest serum sCD14 levels in patients with stable disease compared to patients at the time of an acute relapse or in
Fig. 2. Serum levels of sCD14 in MS patients with acute relapse, stable disease and progression. Data are shown as individual replicates and as boxplot. Medians are indicated by horizontal bars within the boxes.
Fig. 3. Influence of relapses on serum levels of sCD14. Blood samples of MS patients were taken 3 months before an acute relapse (baseline) and at the time of relapse. Data are shown as box-plot. Medians are indicated by horizontal bars within the boxes.
progression. Furthermore, serum sCD14 levels in patients at the time of an acute relapse were lower than in progression. Statistical analysis revealed that these differences in sCD14 levels were not influenced by age, sex, disease duration, EDSS, progression index or MSSS, relapses and MS therapies. The influence of relapses on sCD14 levels was further analyzed in 10 MS patients of whom additional samples taken 3 months before relapse (baseline) were available. In this small group of patients we observed a significant decrease of sCD14 levels from baseline to relapse (Fig. 3). None of the patients in relapse had any clinical or laboratory sign of a current infection. In the whole MS population only 5 (3%) patients had a slightly elevated leukocyte count, without clinical signs of infection. sCD14 levels correlated positively with CRP (r = 0.45, p = 0.003), the percentage of neutrophils (r = 0.27, p = 0.002) and negatively with the percentage of
Fig. 4. Influence of MS therapies on serum levels of sCD14. Blood samples of MS patients treated with IFNβ, other immunomodulatory therapies and untreated MS patients were taken before immunomodulatory therapy was started and one year later. Data are shown as delta sCD14 (1-year baseline). Data are shown as box-plots and medians are indicated by horizontal bars within the boxes.
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lymphocytes (r = −0.27, p = 0.003). There was no correlation between sCD14 and the monocyte count. 3.2. Influence of immunomodulatory treatment on sCD14 serum levels Treatment with immunomodulatory or immunosuppressive drugs had no significant impact on sCD14 serum levels in the overall MS population. However, in a subgroup analysis we found that sCD14 levels were highest in patients with IFNβ treatment (p b 0.05, data not shown). We therefore analyzed the influence of IFNβ treatment on sCD14 serum levels in an independent prospective cohort of 50 MS patients. IFNβ treatment significantly increased sCD14 serum levels in MS patients after one year of treatment compared to baseline (p b 0.001), whereas there was no significant increase of sCD14 levels in patients receiving Glatiramer-acetate, IVIG or patients without therapy (Fig. 4). The change in serum sCD14 levels did not correlate with changes in relapse rate, EDSS, progression index or MSSS. 4. Discussion In this study we report elevated serum levels of sCD14 in patients with MS compared to patients with OND and healthy blood donors. Consistent with our results elevated levels of sCD14 have been found in MS patients and in several autoimmune diseases (Bas et al., 2004; Brettschneider et al., 2002; Egerer et al., 2000; Nockher et al., 1994; Yu et al., 1998). The levels of sCD14 in our healthy controls were comparable to previously published data (Ayaslioglu et al., 2005; Brettschneider et al., 2002; Egerer et al., 2000). Our main finding is that sCD14 levels correlate inversely with the clinical activity of the disease in MS patients. sCD14 levels were significantly increased in MS patients with stable disease compared to patients with an acute relapse or a progressive disease. The difference in sCD14 levels between PPMS and bout onset MS patients might reflect the different inflammatory activity between the two disease courses. Corroborating this result, we observed a decrease in sCD14 levels during relapse in a small number of longitudinally analyzed patients. Thus, the differential expression of sCD14 in MS patients might reflect the current peripheral inflammatory activity of the disease rather than the clinical type of disease course. Brettschneider reported higher sCD14 levels in RRMS compared to PPMS patients without statistical significance between the two groups, but their sample size was considerably smaller (Brettschneider et al., 2002). In the same study they found no difference in sCD14 between patients in relapse and patients in remission. The differences in sCD14 levels observed in our study may also reflect a differential activation and role of the innate immune system in different inflammatory activities of MS. This differential activation of sCD14 might be a result of current infections. In MS patients systemic infections are an important risk factor for exacerbations (Buljevac et al., 2002;
Sibley et al., 1985). Although we did not have any clinical or laboratory evidence of a current infection at the time of relapse in our patients we cannot exclude sub-clinical infections preceding the time point where the blood sample was drawn. The fact that sCD14 levels are elevated in patients during stable disease suggests a protective role of the soluble protein in MS patients. In EAE, CD14 deficient mice have a more severe disease and exhibit a markedly enhanced infiltration of monocytes and neutrophils within the CNS (Walter et al., 2006). In vivo and in vitro studies support the hypothesis that sCD14 has systemic anti-inflammatory effects by reducing the response of monocytes to LPS (Haziot et al., 1994; Kitchens and Thompson, 2005; Kitchens et al., 2001). Furthermore sCD14 has the capacity to negatively regulate T lymphocyte activation and function through direct interaction with T cells and to modulate humoral immune response by interfering with CD40 signaling in B cells (Arias et al., 2000; Rey Nores et al., 1999). In the overall population the correlation of sCD14 with the clinical activity was independent of treatment effect as it was found even in patients without therapy. Brettschneider reported an induction of sCD14 by IFNβ treatment in a serial analysis of PPMS patients (Brettschneider et al., 2002). Therapy with IFNβ for one year induced higher sCD14 serum levels in our RRMS patients as well. It is not clear whether the rise in sCD14 serum levels is due to a direct effect of IFNβ or whether it is an indirect result of a more stable disease course. So far only few data exist on the effect of IFNβ on the expression of the CD14 gene or the soluble protein. A gene expression analysis of IFNβ treated monocytes reported a significant down-regulation of CD14 gene expression in vitro (Wandinger et al., 2001). Differing to this result other groups found that therapy with IFNβ induces a higher expression of CD14 on monocytes and increases the percentage of these CD14 positive cells (Mirowska et al., 2003; Then Bergh et al., 2004). In summary, we provide evidence that serum sCD14 levels and the activation of innate immune response might reflect changes in MS disease activity. References Arias, M.A., Rey Nores, J.E., Vita, N., Stelter, F., Borysiewicz, L.K., Ferrara, P., Labeta, M.O., 2000. Cutting edge: human B cell function is regulated by interaction with soluble CD14: opposite effects on IgG1 and IgE production. J. Immunol. 164, 3480–3486. Ayaslioglu, E., Tekeli, E., Birengel, S., 2005. Significant elevation of serum soluble cd14 levels in patients with brucellosis. Jpn. J. Infect. Dis. 58, 11–14. Bas, S., Gauthier, B.R., Spenato, U., Stingelin, S., Gabay, C., 2004. CD14 is an acute-phase protein. J. Immunol. 172, 4470–4479. Bazil, V., Horejsi, V., Baudys, M., Kristofova, H., Strominger, J.L., Kostka, W., Hilgert, I., 1986. Biochemical characterization of a soluble form of the 53kDa monocyte surface antigen. Eur. J. Immunol. 16, 1583–1589. Brettschneider, J., Ecker, D., Bitsch, A., Bahner, D., Bogumil, T., Dressel, A., Elitok, E., Kitze, B., Poser, S., Weber, F., Tumani, H., 2002. The macrophage activity marker sCD14 is increased in patients with multiple sclerosis and upregulated by interferon beta-1b. J. Neuroimmunol. 133, 193–197.
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