www.elsevier.com/locate/jneuroim

Journal of Neuroimmunology 152 (2004) 191–194

Short communication

Increased blood plasma concentrations of TGF-h isoforms after

treatment with intravenous immunoglobulins (IVIG)

in patients with multiple sclerosis

Dirk Reinholda,*, Evgeniy Perlova, Kirstin Schreckeb, Jorn Kekowc,Thomas Bruned, Michael Sailerb

a Institute of Immunology, Otto-von-Guericke-University Magdeburg, Leipziger Strasse 44, D-39120 Magdeburg, GermanybDepartment of Neurology II, Otto-von-Guericke-University Magdeburg, D-39120 Magdeburg, Germany

cClinic of Rheumatology, Otto-von-Guericke-University Magdeburg, D-39120 Magdeburg, GermanydDepartment of Pediatrics, Otto-von-Guericke-University Magdeburg, D-39120 Magdeburg, Germany

Received 11 November 2003; received in revised form 29 March 2004; accepted 30 March 2004

Abstract

To assess whether TGF-h isoforms are significantly increased after intravenous immunoglobulin (IVIG) infusion in the plasma of patients

with multiple sclerosis (MS), 19 patients with clinically definite MS were enrolled in a double blind placebo controlled IVIG study. TGF-h1,TGF-h2, TGF-h3 plasma concentrations were measured prior and directly after IVIG infusions by specific ELISA.

Compared to the placebo group, we found a significant increase in the plasma levels of all three TGF-h isoforms in patients treated with

IVIG. The significantly increased TGF-h plasma concentrations in treated patients suggest an additional, immediate mechanism of action that

may accompany the molecular effects of IVIG therapy in MS. The variable amount of the potent anti-inflammatory TGF-h isoforms within

the IVIG preparations may exert a differentiated view regarding the manifold indications of IVIG therapy.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Multiple sclerosis; Intravenous immunoglobulins; TGF-h isoforms

1. Introduction

Immunomodulatory treatment constitutes the main ther-

apeutic approach in multiple sclerosis (Fazekas et al.,

1997; Sorensen et al., 2002). Four controlled studies

employing Intravenous immunoglobulins reported a reduc-

tion in relapse rate, progression and evidence of decreased

MRI activity in relapsing-remitting MS (Fazekas et al.,

1997; Achiron et al., 1998a; Sorensen et al., 1998, 2002;

Lewanska et al., 2002). IVIG in MS is currently consid-

ered as an alternative when patients do not tolerate the

approved injected first line medication (Sorensen et al.,

2002).

TGF-h has recently gained increasing consideration from

in vitro an in vivo studies in MS showing suppression of

0165-5728/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.jneuroim.2004.03.018

* Corresponding author. Tel.: +49-391-6715857; fax: +49-391-

6715865.

E-mail address: dirk.reinhold@medizin.uni-magdeburg.de

(D. Reinhold).

inflammatory processes (Racke et al., 1991, 1993). In a

former work, we showed that the multifunctional immuno-

suppressive cytokine TGF-h is present in substantial

amounts in commercially available IVIG preparations

(Kekow et al., 1998; Pap et al., 1998). The aim of this

study is to assess whether different TGF-h isofoms are

significantly increased in the plasma of MS patients after

IVIG-infusions.

2. Material and methods

2.1. Subjects

Twenty-three patients with clinically definite MS were

recruited for the study. Nineteen patients were enrolled in a

double blind placebo controlled IVIG study (SAG-INT10,

Fa. Novartis). Four patients underwent an open label

treatment with IVIG because of contraindications for a

first line immunomodulatory therapy. The treatment dosage

D. Reinhold et al. / Journal of Neuroimmunology 152 (2004) 191–194192

for all patients receiving verum IVIG amounted to 0.4 g

IVIG per kg body weight. The infusion rate was up to 90

ml/h in the first 60 min followed by an infusion rate of

150 ml/h.

All subjects gave written informed consent to enter the

study, which had been approved by the local institutional

review board.

2.2. Blood sample collection

In all patients 10 ml citrate blood samples were col-

lected directly prior and 30 min after the IVIG infusion

and processed without breaking the blinding in the placebo

controlled group. From four patients treated in the open

label mode additionally citrate blood samples were col-

lected at 17, 24, 48, 72 and 96 hours after infusion. The

blood was centrifuged not later than 20 min after sampling

and platelet poor citrate plasma was prepared using a

standardized two-step-separation method (Reinhold et al.,

1997).

2.3. TGF-b1, TGF-b2, and TGF-b3 ELISA

TGF-h1 and TGF-h2 plasma concentrations were mea-

sured with isotype-specific ELISA systems as described

elsewhere (Danielpour, 1993; Szymkowiak et al., 1995).

TGF-h3 plasma concentrations were determined with a new

developed TGF-h3 ELISA, using a goat anti-TGF-h3 and a

monoclonal anti-TGF-h3 antibody (R&D Systems, Minne-

apolis, MN). This assay is sensitive to 20 pg of TGF-h3activity per ml. Samples were tested after transient acidifi-

cation (reduction of the pH to 1.5 by addition of 5 N HCl for

30 min at 37 jC and neutralization with 1.4 N NaOH in 0.7

M Hepes) (Reinhold et al., 1997).

2.4. Statistical analysis

The data were checked against Gaussian distribution and

were expressed as means and standard deviations. Student’s

two-tailed t-test was used to compare the means.

Fig. 1. TGF-h1, TGF-h2, and TGF-h3 concentrations in the plasma of MS

patients before and 30 min after infusion with IVIG and placebo as

measured by specific ELISAs.

3. Results

3.1. Placebo controlled group (19 patients)

The mean concentration of TGF-h1 in the IVIG probes

(n = 11) amounted to 15.2F 2.3 ng/ml, of TGF-h2 to

13.1F 0.8 ng/ml, and of TGF-h3 to 1137F 316 pg/ml,

whereas TGF-h isoforms were not detected in the placebo

(n = 8) preparations.

Fig. 1 shows an increase in the plasma levels of all

three TGF-h isoforms in patients treated with IVIG 30 min

after infusion. The mean plasma concentration of TGF-h1increased from 6.4F 1.8 to 8.4F 1.3 ng/ml ( p< 0.05), of

TGF-h2 from 15.9F 4.75 to 24.1F 9.1 ng/ml ( p < 0.05)

and the concentration of TGF-h3 from 166.0F 59.4 to

362.4F 82.6 pg/ml ( p < 0.05).

In the placebo group no significant changes of TGF-hconcentrations were found (Fig. 1). The mean plasma

Fig. 2. TGF-h1, TGF-h2, and TGF-h3 concentrations in the plasma of four

MS patients before and over a time period of 96 h after infusion with IVIG

as measured by specific ELISAs. Data are presented as meanF SD.

D. Reinhold et al. / Journal of Neuroimmunology 152 (2004) 191–194 193

concentrations prior to infusion were 6.8F 1.6 ng/ml (TGF-

h1), 13.0F 4.2 ng/ml (TGF-h2), and 89.2F 40.2 pg/ml

(TGF-h3) and 6.6F 1.8 ng/ml (TGF-h1), 13.5F 3.2 ng/ml

(TGF-h2), and 82.0F 33.9 pg/ml (TGF-h3) after the infu-

sion (for all comparisons, p>0.05).

3.2. Open label treatment (4 patients)

Citrate blood samples were collected immediately prior

and after 30 min as described above. Additional samples

were collected at 17, 24, 48, 72 and 96 h after infusion. Fig.

2 depicts the TGF-h2 levels that were significantly in-

creased up to 96 h after infusion ( p < 0.05). The concen-

trations of both TGF-h1 and TGF-h3 were found to return

to the pre infusion level after 24 h (Fig. 2).

4. Discussion

Immunomodulatory treatment with IVIG constitutes cur-

rently an accepted therapeutic approach in MS where it

serves as a second line treatment for patients who do not

tolerate the approved injected first line medication (Fazekas

et al., 1997). Modulation of the disease course of MS by

IVIG is achieved by limiting the inflammatory process

whereas the enhancement of remyelination could not have

been proved in vivo as yet. IVIG is discussed to block Fc

receptors in mononuclear phagocytes, to suppress both

MHC antigen presentation and antigen recognition by the

T cell receptor, to decrease the production of disease-

promoting cytokines like TNF-a and IFN-g, and to block

autoantibodies (Achiron et al., 1998b). The data presented

here propose a supplementary way of action beside the

already known molecular mechanisms. We found in MS

patients treated with IVIG a significant increase in plasma

concentration of all TGF-h isoforms after infusion particu-

larly a significant increased TGF-h2 plasma concentrations

up to 96 hours after infusion.

Despite the rapidly degrading properties of the TGF-hisoforms in vivo, the substantial amounts of the presence of

this potent anti-inflammatory cytokine found in the plasma

of patients after treatment with IVIG may suggest an

immediate initiation or at least enhancement of the immu-

nosuppressive cascade.

In the in vivo model of multiple sclerosis the experimen-

tal autoimmune encephalomyelitis (EAE) it has been

demonstarted that treatments with IVIG as well as with

TGF-h1 or TGF-h2 are capable of preventing EAE and

suppressing an already established disease (Pashov et al.,

1997, 1998; Piccirillo and Prud’homme, 1999, Racke et al.,

1991, 1993).

While the immunosuppressive effect of TGF-h in the

therapy of T cell-mediated autoimmune diseases like MS

would have to be considered as an advantage, in supple-

mentary therapies with the aim to support the immune

response it may exert a negative effect. IVIG treatment is

also the therapy of choice in patients with innate immune

deficiencies like Bruton’s diseases, SCID, Wiskott-Aldrich-

Syndrome or MHC II-deficiency IVIG (Dwyer, 1992;

Schiff, 1994a,b). Moreover, IVIG is recommended in the

therapy of neonatal sepsis (Jenson and Pollock, 1997). Due

to the well-characterized immunosuppressive effects of

TGF-h in vitro and in vivo, in these indications the presence

of high amounts of TGF-h in IVIG preparations would

represent a great disadvantage.

In conclusion, the understanding of the application of

IVIG in the rapidly growing ‘‘off label’’ therapeutic field is

crucial in order to constitute appropriate treatment regimes.

This seems even more important in the light of the complex

mechanism of action of IVIG and the possibility that

variable ‘‘by-products’’ like TGF-h could be responsible

for some of the therapeutic effects.

Acknowledgements

We thank K. Mnich, K. Barthels and B. Schultze for their

excellent technical help.

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