Journal of Neuroimmunology 200 (2008) 129–132www.elsevier.com/locate/jneuroim

Short communication

The expression of TNF-α receptors 1 and 2 on peripheral blood mononuclearcells in chronic inflammatory demyelinating polyneuropathy

Xin Yang a,b,1, Tao Jin a,b,1, Rayomand Press c, Hernan Concha Quezada d,Sten Fredrikson c, Jie Zhu a,b,⁎

a Division of Neurodegeneration, Department of Neurobiology, Care Sciences and Society, Karolinska Institute, Karolinska University Hospital Huddinge,Stockholm, Sweden

b Department of Neurology, the First Hospital, Jilin University, Changchun, Chinac Section of Neurology, Department of Clinical Neuroscience, Karolinska Institute, Karolinska University Hospital Huddinge, Stockholm, Swedend Center for Infectious Medicine, Department of Medicine, Karolinska Institute, Karolinska University Hospital Huddinge, Stockholm, Sweden

Received 16 February 2008; received in revised form 30 May 2008; accepted 4 June 2008

Abstract

The proportion of peripheral blood mononuclear cells (PBMCs) expressing TNF-α and its receptors (TNFR1, TNFR2) and the serumconcentrations of its soluble forms were analyzed by FACS and ELISA in the patients with chronic inflammatory demyelinating polyneuropathy(CIDP) and in controls. Elevated levels of TNFR2 were observed on blood T cells in CIDP and idiopathic polyneuropathy. Low levels of TNFR1were detected on monocytes in the subgroup of patients with CIDP examined after treatment with intravenous immunoglobulin. However, theproliferative activity of PBMCs in CIDP was not influenced by soluble recombinant TNFR1. Our limited data suggested the exact role of TNF-αand its receptors need to study further in CIDP, as well as in idiopathic neuropathies.© 2008 Elsevier B.V. All rights reserved.

Keywords: Chronic inflammatory demyelinating polyneuropathy (CIDP); Idiopathic polyneuropathy (PNP); Tumor necrosis factor (TNF-α); Receptors; Immunoglobulin

1. Introduction

Tumor necrosis factor (TNF-α) is generally considered as amultifunctional proinflammatory cytokine which can mediatechronic inflammatory pathology and tissue damage in chronicinflammatory demyelinating polyneuropathy (CIDP) (Kieseieret al., 2002). However, the findings indicateing that differentTNF receptors exert opposite effects on inflammatory path-ways. TNFR1 (p55) has been shown to be a crucial mediator ofmyelin glycoprotein-induced experimental allergic encephalo-myelitis in TNFRs knockout mice (Myers et al., 2006), whilethe TNFR2 (p75) signaling has a protective role in the clinical

⁎ Corresponding author. Division of Neurodegeneration (NOVUM, plan 5),Department of Neurobiology, Care Sciences and Society, Karolinska Institute,Karolinska University Hospital in Huddinge, Stockholm, Sweden, SE-141 86Stockholm, Sweden. Tel.: +46 8 58585494; fax: +46 8 58585470.

E-mail address: Jie.Zhu@ki.se (J. Zhu).1 These authors contributed equally to the work.

0165-5728/$ - see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.jneuroim.2008.06.003

progression of the Guillain–Barré syndrome (GBS) (Radhak-rishnan et al., 2004). These findings make the exact pathogenicrole of TNF-α in CIDP controversial.

In this study, we analyzed the proportion of peripheral bloodmononuclear cells (PBMCs) expressing TNF-α and TNFRs,and the serum concentrations of its soluble forms in the pa-tients with CIDP and in controls. Additionally, the role of PEGr-metHu-sTNFR1, a soluble TNF-α antagonist which competesfor the ligands with membrane-bound TNFRs, was analyzed byPBMCs proliferation assay with the aim to evaluate analternative therapeutic approach in CIDP.

2. Materials and methods

2.1. Patients

The study population comprised 11 patients with CIDP(Table 1). All patients with CIDP had a progressive motor and

Table 1Survey of the clinical features of CIDP, PNP and OND patients

Characteristics CIDP (n=11) PNP (n=11) OND (n=11)

Age (years, mean±SD) 60.64±4.66 67.54±3.14 63.91±10.23Male/female 8/3 8/3 8/3Duration (years, mean±SD) 8±6.12 19.27±21.91Clinical signs, M/S/MS/SM 5/0/5/1 0/2/5/4Symptoms, pain/painless 2/9 7/4Neurophysiological findings, predominatelydemyelinating/predominately axonal/mixed

8/1/2 0/3/8

Treatment, CS/IvIg/untreated 1/6/4 0/0/11Concurrent disease 3 diabetes; 2 previous GBS; 1 cardiac arrhythmia;

1 psoriasis; 1 psychosis1 ulcerative colitis; 1IgG paraprotein;1 psoriasis

1 hypertension

CIDP chronic inflammatory demyelinating polyneuropathy; PNP = idiopathic polyneuropathy; OND = other neurological diseases; M = motor symptoms; S = sensorysymptoms; MS = motor symptoms > sensory symptoms; SM = sensory symptoms > motor symptoms; CS = corticosteroids (Prednisone, oral treatment); IvIg =intravenous high dose immunoglobulin (at a dosage of 0.4g/kg/d for 5 days); IgG = immunoglobulin G.

130 X. Yang et al. / Journal of Neuroimmunology 200 (2008) 129–132

sensory neuropathy. The CIDP diagnosis was based on the Amer-ican Academy of Neurology AIDS Task Force criteria (Ad HocSubcommittee for the American Academy of Neurology AIDSTask Force, 1991). Four of 11 patients with CIDP were examinedbefore initiation of immunomodulatoroy treatment, six patientswere examined already on treatment with high dose of intravenousimmunoglobin (IvIg), and one while on corticosteroids.

Control patients comprised of those with idiopathic polyneu-ropathy (PNP), other neurological diseases (OND) and healthycontrols. Idiopathic PNP was defined as a predominantly axonalneuropathy, where definite etiological factors such as systemicmetabolic disorders, toxic agents etc. were excluded (Table 1).The OND consisted of patients with amyotrophic lateral scle-rosis (5 patients), epilepsy (1 patient) and Parkinson disease(5 patients). Eight healthy blood donors were also included ashealthy controls in this study (Table 1).

Blood from all the patients mentioned above subsequentlyunderwent flow cytometric analysis and PBMCs proliferation.Serum from 8 patients with CIDP, 10 patients with PNP, 10patients with OND and 5 healthy controls were examined by theenzyme linked immunosorbent assays (ELISA).

This project was approved by the local ethical committee atStockholm, Sweden.

2.2. PBMCs preparation and flow cytometric analysis

PBMCs were prepared as previously described (Luet al., 2007). Serum samples were also collected and

Table 2The levels of TNFR1 and TNFR2 expression on the circulating T cells, B cells and

Groups TNFR1 (positive cells, %)

CD3 CD19 CD14

CIDP 0.23 (0.18–0.70) 0.03 (0.02–0.38) 2.38 (1.05–2.65PNP 0.36 (0.12–0.68) 0.07 (0.02–0.19) 1.19 (0.61–2.07OND 0.51 (0.14–0.70) 0.17(0.07–0.30) 1.26 (0.58–2.19Healthy controls 0.13 (0.12–0.18) 0.05 (0.01–0.09) 1.48 (0.17–2.12

Data are stated as median values and (first and third quartiles). Values compared to⁎⁎Pb0.01).CIDP chronic inflammatory demyelinating polyneuropathy; PNP = idiopathic polyTNFR2 = TNF-α receptor 2.

cryopreserved at −80 °C for further ELISA. Regarding two-color flow cytometry, PE-conjugated monoclonal antibody(mAb) specific for TNFR1 (Serotec, Oxford, UK) and TNFR2(Caltag Laboratories, Burlingame, California, USA), FITC-conjugated mAb anti-CD3, anti-CD19 and CD14 (BD Bios-ciences Pharmingen, San Jose, CA) were used with thecorresponding isotype controls as previously described (Luet al., 2007).

2.3. Serum measurements of soluble TNF-α and its receptors

To determine the levels of TNF-α (Diaclon, Becancon,France), soluble TNFR1 and TNFR2 (Bender MedSystem Inc.,Vienna, Austria), ELISAwas performed according to the manu-facture's instructions. The serum was diluted 1:3 for TNFR1and 1:2 for TNFR2 detection. If concentrations of TNF-α wereb12.5 pg/ml, the values were replaced by 12.5 pg/ml. All assayswere done in duplicate.

2.4. PBMCs proliferation with PEG r-metHu-sTNFR1

PBMCs from patients with CIDP, PNP, OND and healthycontrols were cultured in vitro in the presence of PEG r-metHu-sTNFR1 (final concentration: 7.5 μg/ml; Amgenand Wyeth Pharmaceutical Inc. CA, USA) for 48 hand labeled as previously described (Lu et al., 2007).Cultures were run in triplicates and the results wereexpressed as cpm.

monocytes by flow cytometric analysis

TNFR2 (positive cells, %)

CD3 CD19 CD14

) 27.69 (23.34–31.52)⁎ 0.26 (0.12–0.84) 61.14 (13.52–88.65)) 28.7 (24.32–34.72)⁎⁎ 0.33 (0.16–0.44) 84.38 (35.37–94.76)⁎

) 19.34 (16.82–34.72) 0.83 (0.30–1.44) 81.33 (58.35–89.53)⁎

) 18.66 (3.44–22.48) 0.13 (0.03–0.25) 55.87 (1.90–60.68)

the corresponding level of healthy control, difference is significant (⁎Pb0.05,

neuropathy; OND = other neurological diseases; TNFR1 = TNF-α receptor 1;

Fig. 1. The levels of TNFR1 expression on circulating monocytes in untreatedand treated patients with CIDP. The level of TNFR1 expression on circulatingmonocytes of CIDP patients (4 untreated and 7 treated) vs. 8 healthy controls(HC). Each simple box plot shows the median and quartiles. ⁎Pb0.05, N.S.means no significance. CIDP = chronic inflammatory demyelinating poly-neuropathy; TNFR1 = TNF-α receptor 1.

Fig. 2. Concentration of sTNFR1 in serum (ng/ml) in patients with CIDP andcontrols. The concentration of soluble TNFR1 in serum of patients with CIDP(n=8), PNP (n=10), OND (n=10) and healthy controls (HC, n=5). The dashedline represents 3 SD above the mean value of healthy controls. CIDP chronicinflammatory demyelinating polyneuropathy; PNP = idiopathic polyneuro-pathy; OND = other neurological diseases; sTNFR1 = soluble TNF-α receptor 1in serum.

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2.5. Statistical analysis

Differences of median values were tested with the Kruska–Wallis nonparametric test for multiple comparisons and thenonparametric Mann–Whitney U test for two groups, usingSPSS software (version 11.15). Statistical significance was ac-cepted at Pb0.05. Spearman rank test was used for correlationanalysis.

3. Results

3.1. Increased expression of TNFR2 on blood T cells in CIDPand PNP

Similar to the finding by Beltinger et al. (1996), TNFR2 wasfound as the main TNF-α receptor on circulating T cells andmonocytes in all the examined patients (Table 2). On the con-trary, neither TNFR1 nor TNFR2 was expressed highly oncirculating B cells.

Table 3Levels of TNF-α and soluble TNF receptors of patients with CIDP, PNP, ONDand healthy controls by ELISA

Groups TNF-α (pg/ml) sTNFR1 (ng/ml) sTNFR2 (ng/ml)

CIDP 12.50 (12.50–12.50) 0.25 (0.22–0.36) 1.67 (0.92–2.52)PNP 12.50 (12.50–12.50) 0.23 (0.21–0.43) 1.63 (0.54–2.91)OND 12.50 (12.50–28.71) 0.31 (0.22–0.44) 2.01 (0.90–4.32)Healthy controls 12.50 (12.50–55.87) 0.24 (0.21–0.28) 1.36 (0.57–3.12)

Data are stated as median values and (first and third quartiles).CIDP chronic inflammatory demyelinating polyneuropathy; PNP = idiopathicpolyneuropathy; OND = other neurological diseases; TNF-α = tumor necrosisfactor; sTNFR1 = soluble TNF-α receptor 1 in serum; TNFR2 = soluble TNF-αreceptor 2 in serum.

As compared with healthy controls, there was a significantelevation of TNFR2 expression on circulating T cells in both theCIDP (Pb0.05) and PNP groups (Pb0.01), but no differenceexisted between the two groups (Table 2). The expression ofTNFR2 on blood monocytes was higher in patients with PNPand OND (Pb0.05), but not in patients with CIDP (Table 2).The expression of TNFR1 on T cells and monocytes were notelevated in patients with CIDP, PNP and OND.

Fig. 3. Ratio of cpm value between baseline and soluble TNFR1 during in vitrotreatment of PBMC from patients with CIDP and controls. The ratio (after treatedwith PEG r-metHu-sTNFR1: baseline without any treatment) of CIDP (n=11),PNP (n=11), OND (n=11) patients and healthy controls (HC, n=8) were shownin y-axis. The arrows refer to the median value in each group. There was nosignificance between any two groups. N.S. means no significance. CIDP chronicinflammatory demyelinating polyneuropathy; PNP = idiopathic Polyneuropathy;OND = Other Neurological Diseases; TNFR1 = TNF-α Receptor 1.

132 X. Yang et al. / Journal of Neuroimmunology 200 (2008) 129–132

3.2. The levels of TNFR1 expression on circulating monocyteswas lower in patients with CIDP

The patients with CIDP were subdivided into those ex-amined prior to receiving immunomodulatory treatment (n=4),and those examined 4–5 weeks after a course of IvIg treatment(n=6). The levels of TNFR1 expression on monocytes weresignificantly (Pb0.05) lower in patients with CIDP examinedafter initiation of treatment with IvIg vs. healthy controls (Pb0.05), while levels of TNFR1 expression on blood monocytesin untreated CIDP did not differ from healthy controls (Fig. 1).

The levels of TNFR1 on circulating T and B cells were notaltered in CIDP examined prior to (n=4) vs. following treat-ment with IvIg (n=6).

3.3. Serum measurements and the in vitro therapeutic effect ofsoluble TNFR1

The cutoff values of TNF-α, TNFR1 and TNFR2 were set to114.34 pg/ml, 1.44 ng/ml and 18.65 ng/ml according to the fivehealthy controls (mean±3 SD) (Table 3). Using these cutoffvalues, elevated serum levels of soluble TNFR1 were detectedin two out of 8 patients with CIDP, two out of 10 patients withPNP and three out of 10 patients with OND (Fig. 2, Table 3).

The effect of soluble TNFR1 on PBMCs was defined as theratio between cpm values of the baseline to post-treatment withsoluble TNFR1. Subsequently, different ratios were comparedin patients with CIDP (n=11), PNP (n=11), OND (n=11)and healthy controls (n=8). There was no significant differencefound between these groups (Fig. 3).

4. Discussion

Two distinct TNFRs, both existing as cell-surface mem-brane-bound and soluble form, are believed to play differentroles in the biological activities of TNF-α (Myers et al., 2006).Generally, the classic proinflammatory activities of TNF-α,which account for chronic inflammatory pathology, are me-diated by TNFR1 (Barbara et al., 1994; Pfeffer, 2003), whileTNFR2 is considered to have an anti-inflammatory and neu-roprotective function (Kollias and Kontoyiannis, 2002; Myerset al., 2006). The significance of increased levels of TNFR2expression on blood T cells in patients with inflammatory-as well as non-inflammatory neuropathies examined in thisstudy is not clear. TNFR2 has been shown to have an anti-inflammatory role in many autoimmune models (Dopp et al.,1997; Peschon et al., 1998; Myers et al., 2006). TNFR2 ex-pressing blood T cells may thus have a protective role againstprimary and or secondary autoimmunity targeted towards pe-ripheral nerve antigens in CIDP and idiopathic neuropathies.

Another finding of this study was the decrease of TNFR1expression on blood monocytes in patients with CIDP who wereexamined after immunomodulatory treatment, despite normallevels of TNFR1 expression prior to treatment. This phenom-enon may be interpreted to merely reflect a disease-unrelated

effect of IvIg on immune blood cells, rather than an explanationor a disease-promoting role for TNFR1 in CIDP.

Unlike previous reports of elevated levels of TNF-α in serumof patients with CIDP (Empl et al., 2001), we found no increasein levels of soluble TNF-α in CIDP. Finally, our study showsthat the soluble recombinant TNFR1, PEG r-metHu-sTNFR1had no effect on in vitro proliferation of PBMCs from patientswith CIDP, suggesting that blocking the biological action ofTNF-α, may not be useful as a therapeutic method in CIDP.

Our limited data indicates that the exact roles of TNF-α andits receptors (mainly TNFR2) warrant further studies in thepathogenesis of peripheral inflammatory- as well as non-inflammatory neuropathies.

Acknowledgements

ALF-project grant from Karolinska Institute UniversityHospital, Stockholm, Sweden. We sincerely thank G.X. Jiang,senior researcher, at Dept. Psychological Medicine, KarolinskaInstitute for statistical analysis.

References

Ad Hoc Subcommittee for the American Academy of Neurology AIDS TaskForce, 1991. Research criteria for chronic inflammatory demyelinatingpolyneuropathy (CIDP). Neurology 41, 617–618.

Barbara, J.A., Smith, W.B., Gamble, J.R., 1994. Dissociation of TNF-alphacytotoxic and proinflammatory activities by p55 receptor- and p75 receptor-selective TNF-alpha mutants. EMBO J. 13, 843–850.

Beltinger, C.P., White, P.S., Maris, J.M., Sulman, E.P., Jensen, S.J., LePaslier,D., Stallard, B.J., Goeddel, D.V., de Sauvage, F.J., Brodeur, G.M., 1996.Physical mapping and genomic structure of the human TNFR2 gene.Genomics 35, 94–100.

Dopp, J.M.,Mackenzie-Graham,A., Otero, G.C.,Merrill, J.E., 1997. Differentialexpression, cytokine modulation, and specific functions of type-1 and type-2tumor necrosis factor receptors in rat glia. J. Neuroimmunol. 75, 104–112.

Empl, M., Renaud, S., Erne, B., Fuhr, P., Straube, A., Schaeren-Wiemers, N.,Steck, A.J., 2001. TNF-alpha expression in painful and nonpainfulneuropathies. Neurology 56, 1371–1377.

Kieseier, B.C., Dalakas,M.C., Hartung, H.P., 2002. Immunemechanisms in chronicinflammatory demyelinating neuropathy. Neurology 59 (12 Suppl 6), S7–12.

Kollias, G., Kontoyiannis, D., 2002. Role of TNF/TNFR in autoimmunity:specific TNF receptor blockade may be advantageous to anti-TNFtreatments. Cytokine Growth Factor Rev. 13, 315–321.

Lu, M.O., Duan, R.S., Quezada, H.C., Chen, Z.G., Mix, E., Jin, T., Yang, X.,Ljunggren, H.G., Zhu, J., 2007. Aggravation of experimental autoimmuneneuritis in TNF-alpha receptor 1 deficient mice. J. Neuroimmunol. 186, 19–26.

Myers, R.R., Campana, W.M., Shubayev, V.I., 2006. The role of neuroin-flammationin neuropathic pain: mechanisms and therapeutic targets. DrugDiscov. Today 11, 8–20.

Peschon, J.J., Torrance, D.S., Stocking, K.L., Glaccum, M.B., Otten, C., Willis,C.R., Charrier, K., Morrissey, P.J., Ware, C.B., Mohler, K.M., 1998. TNFreceptor-deficient mice reveal divergent roles for p55 and p75 in severalmodels of inflammation. J. Immunol. 160, 943–952.

Pfeffer, K., 2003. Biological functions of tumor necrosis factor cytokines andtheir receptors. Cytokine Growth Factor Rev. 14, 185–191.

Radhakrishnan, V.V., Sumi, M.G., Reuben, S., Mathai, A., Nair, M.D., 2004.Serum tumour necrosis factor-alpha and soluble tumour necrosis factorreceptors levels in patients with Guillain–Barre syndrome. Acta Neurol.Scand. 109, 71–74.