Expression of Antigen Processing and Presenting Mollecules by Schwann Cell in Inflamatory Neuropathy

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Glia. Author manuscript; available in PMC 2012 October 25.
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Glia. 2010 January 1; 58(1): 8092. doi:10.1002/glia.20903.
Expression of Antigen Processing and Presenting Molecules by

Schwann Cells in Inflammatory Neuropathies
GERD MEYER ZU HORSTE1,*, HOLGER HEIDENREICH1, HELMAR C. LEHMANN1,
SOLDANO FERRONE2, HANS-PETER HARTUNG1, HEINZ WIENDL3, and BERND C.
KIESEIER1
1Department of Neurology, Research Group for Clinical and Experimental Neuroimmunology,
Heinrich-Heine-University, Dsseldorf, Germany

2Departments of Surgery, Immunology, and Pathology, University of Pittsburgh Cancer Institute,
Pittsburgh, Pennsylvania
3Department of Neurology, Julius-Maximilians-University, Wrzburg, Germany
Abstract
Schwann cells are the myelinating glia cells of the peripheral nervous system (PNS) and can
become targets of an autoimmune response in inflammatory neuropathies like the Guillain-Barr
syndrome (GBS). Professional antigen presenting cells (APCs) are known to promote autoimmune
responses in target tissues by presenting self-antigens. Other cell types could participate in local
autoimmune responses by acting as nonprofessional APCs. Using a combined approach of
immunocytochemistry, immunohistochemistry, and flow cytometry analysis we demonstrate that
human Schwann cells express the antigen processing and presenting machinery (APM) in vitro
and in vivo. Moreover, cultured human Schwann cells increase the expression of proteasome
subunit delta (Y), antigen peptide transporter TAP2, and HLA Class I and HLA Class II
complexes in an inflammatory environment. In correlation with this observation, Schwann cells in
sural nerve biopsies from GBS patients show increased expression of antigen processing and
presenting molecules. Furthermore, cultured human Schwann cells can proteolytically digest
fluorescently-labeled nonmammalian antigen ovalbumin. Taken together, our data suggest antigen
processing and presentation as a possible function of Schwann cells that may contribute to
(auto)immune responses within peripheral nerves.
Keywords
inflammatory neuropathy; Guillain-Barr syndrome; Schwann cell; antigen presentation
INTRODUCTION
Immune-mediated neuropathies, such as the acute Guillain-Barr syndrome (GBS), result
from an autoimmune response within the peripheral nervous system (PNS) (Griffin and
Sheikh, 2005; Hughes and Cornblath, 2005). In the more common demyelinating subtypes
of GBS the autoimmune response is considered to be directed against Schwann cell rather
than axonal components (Kieseier et al., 2004), although the target antigen remains elusive.
2009 Wiley-Liss, Inc.

*
Correspondence to: Gerd Meyer zu Horste, Department of Neurology, Heinrich-Heine-University, Moorenstrasse 5, 40225
Dsseldorf, Germany. gerd.mzh@uni-duesseldorf.de.
Additional Supporting Information may be found in the online version of this article.
HORSTE et al. Page 2
Putative autoreactive T lymphocytes are under continuous control to ensure self-tolerance

but may be activated during minor infections by the encounter of microbial epitopes
resembling endogenous peripheral nerve antigens. In the antibody-mediated axonal subtypes
of GBS, such cross-reactivity of anti-microbial antibodies towards endogenous nerve-

derived epitopes, has been demonstrated and termed molecular mimicry (Willison and Yuki,
2002; Yuki, 2005). Apart from antibodies and complement, both CD4+ and CD8+
subpopulations of T lymphocytes infiltrate into peripheral nerves in GBS and may thus
participate in the immune-mediated damage (Fujioka et al., 2000; Pollard et al., 1987;
Schmidt et al., 1996). To which extent humoral and cellular immune components mediate
tissue damage in demyelinating subtypes of GBS remains subject to debate.
Peptide antigens are presented in the context of major histocompatibility complex (MHC)
molecules from antigen presenting cells (APCs) to T cells (Trombetta and Mellman, 2005)
enabling antigen-specific responses of the adaptive immune system. In the classical concept
of antigen presentation most nucleated human cells express and utilize human lymphocyte
antigen (HLA) Class I molecules to present antigenic peptides from endogenous sources to
CD8+ cytotoxic T lymphocytes. Only professional APCs, like dendritic cells and
macrophages, present peptides generated from foreign exogenous proteins on HLA Class II
molecules that are recognized by CD4+ helper T cells (Trombetta and Mellman, 2005).
Several aspects of this concept, however, have had to be revised. The intracellular
processing pathways of antigenic peptides are not strictly separated. In some settings Class I
molecules present internalized antigen (Pfeifer et al., 1993), a mechanism referred to as
cross-presentation (Ackerman and Cresswell, 2004). Endogenously synthesized cytosolic
antigens can also be presented on HLA Class II molecules (Dani et al., 2004; Malnati et al.,
1992; Weiss and Bogen, 1991). Furthermore, different cell typese.g. endothelial cells
apart from professional APCs may express HLA Class II molecules following inflammatory
stimuli (Bottazzo et al., 1983; Choi et al., 2004). Presentation of antigenic peptides on HLA
Class I and II molecules on such nonprofessional or facultative APCs may locally promote
or perpetuate a systemically initiated immune response. Knowledge on antigen processing
and presentation capabilities of nonprofessional antigen presenting cell types in the
peripheral nerve is therefore crucial to the understanding of pathogenetic events in immune
neuropathies.
Schwann cells represent interesting candidates for such facultative APCs. We here provide
evidence that human Schwann cells contain the elements required to process and present
endogenous and exogenous peptides to T lymphocytes (antigen processing and presenting
machinery (APM)) and provide functional evidence for such a potential disease mechanism.
Elucidating Schwann cell-lymphocyte interactions may offer interesting novel therapeutic
targets for modulating clinical disease manifestations in inflammatory neuropathies in the

future.
METHODS
Schwann Cell Culture
Primary human Schwann cells isolated from early postnatal human tissue were purchased
from ScienCell (San Diego, CA) and cultured following standard protocols (Casella et al.,
1996; Rutkowski et al., 1995). Briefly, human Schwann cells were plated on poly-D-lysine
coated plastic dishes and maintained in basal medium consisting of Dulbeccos modified
Eagles medium (DMEM) supplemented with 10% fetal calf serum, 100 U mL1 penicillin,
100 g mL1 streptomycin, 2 mM glutamine, and 2 M forskolin (all from Gibco-
Invitrogen, Karlsruhe, Germany). Cells were kept at 37C in a 10% CO2 humidified
atmosphere and proliferated well until passage 14 to 15 and then gradually decreased growth

until no further culturing was possible at approximately passage 16. Schwann cell identity
and purity was above 98% at passages three, five, nine, and twelve as determined by S100
staining.
Processing of DQ-Ovalbumin by Cultured Human Schwann Cells

Human monocytes were isolated from blood of healthy human donors using antibody-based
negative selection followed by density medium gradient centrifugation following the
manufacturers protocol (Rosette-Sep, StemCell Technologies, Vancouver, BC, Canada) and
cultured under established basal conditions. DQ-Ovalbumin (25 g mL1, Molecular
Probes-Invitrogen, Karlsruhe, Germany) was added simultaneously without any detergents
to human primary Schwann cells cultured at passage seven at ~80% confluence and human
monocytes, which were both maintained under basal conditions as described above.
Following incubation with DQ-Ovalbumin for 1/2, 1, 3, 6, 12, and 24 h, individual wells
were washed twice with PBS and analyzed for green fluorescence by microscopy. Triplicate
wells of Schwann cells and monocytes were analyzed for green fluorescence by flow
cytometry (FITC channel). Gating excluded dead cells and flow cytometry settings were
kept identical for both cell types and at all time points analyzed.
Immunocytochemistry
Human Schwann cells were fixed for 3 min in 100% acetone at 20C, blocked for 1 h with
10% bovine serum albumin (BSA; Roth, Karlsruhe, Germany) and incubated with primary
antibodies for 1 h (summarized in Table 1). Cells were subsequently incubated for 1 h with
secondary TRITC labeled anti-mouse IgG antibody (1:100), rabbit anti-S100 antibody
(1:100), and Alexa-fluor-488 labeled anti-rabbit IgG antibody (1.5 mg mL1 diluted to
1:500). All antibodies were diluted in 1% Triton in PBS. Mounting medium contained 4,6-
Diamidino-2-phenylindol (DAPI) for nuclear staining. Fluorescent staining intensity was
assessed by visual rating.
Flow Cytometry of Schwann Cells

Cultured human Schwann cells were cultured in the presence or absence of recombinant
human IFN- (500 U mL1), interleukin (IL)-2 (50 U mL1), and IL-4 (8 ng mL1) (all
reagents from Sigma-Aldrich) for 48 h at 37C. Afterwards cells were intracellularly stained
with APM recognizing antibodies (Table 1) as previously described (Ogino et al., 2003).
Simultaneously, Schwann cell samples were incubated with propidium iodide (PI) at 1 g
mL1 in PBS for 5 min and subsequently analyzed by flow cytometry. Gating excluded dead
cells by forward and sideward scatter and previously PI confirmed viability in the gate
applied.
Human Nerve Biopsies

Sural nerve biopsies were obtained for diagnostic purposes with informed consent from
patients as previously described (Hu et al., 2003). Two groups of patients were analyzed.
The first group included five patients diagnosed as GBS according to the criteria of Asbury
and Cornblath (1990) with median disease duration before biopsy of 8 days ranging from 3
to 18 days (Table 2). All patients were diagnosed as the acute inflammatory demyelinating
polyneuropathy (AIDP) subtype of GBS. None of the patients with GBS had received any
immunomodulatory or immunosuppressive treatment within 3 months before biopsy.
Noninflammatory controls (n = 5) included one healthy subject, one case of radiculitis, two
cases of hereditary neuropathy with liability to pressure palsies without obvious
impairments, and one case of toxic neuropathy (Table 2). Nerve biopsies were formalin-
fixed and paraffin embedded.

Immunohistochemistry
Paraffin-embedded nerve biopsies were cut to 5-m sections on a standard microtome
(HM355S, Microm, Walldorf, Germany) and stained as previously described (Hu et al.,
2003). Briefly, tissue sections were blocked with 10% BSA for 1 h and incubated with
primary antibodies at 4C overnight (Table 1). For fluorescent staining the sections were
further incubated with fluorescently-labeled anti-mouse secondary antibodies (1:100) for 1 h
(Table 1), followed by an S100 antibody (1:100) for 2 h, and a fluorescently labeled anti-
rabbit antibody (1:100) for 1 hour (Table 1). Sections were incubated with DAPI for 5 s,
mounted in 80% glycerol in PBS (Aquatex, Merck, Darmstadt, Germany) and were
photographed on the subsequent day. For nonfluorescent staining and for the localization of
inflammatory infiltrates the staining protocol was alternatively continued by incubating the
sections in 3% H2O2 in methanol for 20 min. A biotinylated secondary antibody was added
for 1 h followed by an avidin-biotin-horseradish peroxidase complex (DAB Kit, DAKO,
Hamburg, Germany). The 3,3-diaminobenzidine (DAB) was added as peroxidase substrate
according to manufacturers instructions. Between all protocol steps sections were washed
for 5 min in PBS. Slides were dehydrated and mounted in xylene-based medium (Entelan,
Merck). All incubations were performed at room temperature unless indicated otherwise.
Analysis of Staining Intensity

Immunohistochemical staining intensity was quantified as a semi-quantitative measure of
expression in Schwann cells of the sural nerve. For this purpose nerve sections from
noninflammatory controls (n = 5) and GBS patients (n = 5) were stained with APM-specific
antibodies (Table 1) as described above without counter-staining. The DAB protocol was
modified by freshly adding nickel-II-chloride (500 g mL1; Merck) to the DAB buffer to
obtain gray DAB staining. Staining of all nerve sections was performed in parallel for
each antibody. Serial tissue sections immediately adjacent (5 m) to the ones used for
quantification were fluorescently labeled with S100 antibody and DAPI as described above
and the number of S100 positive nuclei was counted in the areas used for quantification.
Areas containing S100 negative cells were selected to be excluded from staining intensity
analysis. Five grayscale photographs of each tissue section were taken. Photographs from all
samples were taken without interruption and using identical microscope settings. The mean
gray value was measured in areas containing no artifacts and no S100 negative cells using
ImageJ (v1.36b, NIH, Bethesda, MD) as previously described (Matkowskyj et al., 2000,
2003). Gray values representing the darkness of the area ranging from 0 (white) to 255
(black) were averaged between five photographs per nerve section and compared between
the GBS and the control group. The observer (G.M.z.H.) was blinded towards sample
designation when photographing and analyzing the tissue sections.
Data Acquisition and Analysis

Microscopic slides were analyzed and photographed using a conventional fluorescence
microscope (Axioplan 2, Carl Zeiss, Gttingen, Germany) and cell cultures were analyzed
using an inverted fluorescence microscope (Nikon Eclipse TE200, Nikon, Dusseldorf,
Germany). Flow cytometry was performed using a FACSCantoII flow cytometer (Becton
Dickinson, Heidelberg, Germany) and all flow cytometry data analyses were performed
using FlowJo software (v7.2.5, Treestar, Ashland, OR, USA). Data analysis was performed
using GraphPad Prism 5.0 (GraphPad Software, San Diego California USA). Students t test
for unrelated samples was applied to test for significant differences of staining intensity and
data were analyzed for correlation using Spearmans rank correlation test.

RESULTS
Human Schwann Cells Express Main Components of the Antigen Processing and
Presenting Machinery at Basal Conditions In Vitro

Primary human Schwann cells were maintained in culture and confirmed to specifically
express Schwann cell markers S100 (Fig. 1, left column of panels), neurotrophin receptor
p75 (Supp. Info. Fig. 1A), and low levels of glial fibrillary acidic protein (GFAP; Supp.
Info. Fig. 1B). Visual assessment of fluorescent staining of unstimulated human Schwann
cells demonstrated weak staining for the constitutive proteasome subunits MB1 (X) and Z
(not shown), and strong staining for subunit delta (Y) (Fig. 1A). Among the
immunoproteasome subunitstermed low molecular mass proteins (LMP)LMP2 showed
moderate staining (Fig. 1B), while LMP7 and LMP10 did not return any signal (not shown).
Antigenic peptides are transported to the endoplasmatic reticulum (ER) by the transporter
associated with antigen presentation (TAP). In our Schwann cells cultures we found weak
staining intensity for TAP1 and strong staining for TAP2 (Fig. 1C). Tapasinessential
component of the HLA peptide loading complex of the ERreturned moderate signal (Fig.
1D). Among the ER chaperones we found expression of calnexin (Fig. 1E) and no
immunoreactivity for calreticulin (not shown). By immunocytochemistry HLA Class I
molecules were clearly expressed (Fig. 1F) and we observed staining intracellularly as well
as on the cell membrane. Our Schwann cell cultures also stained weakly positive for HLA-
DR,-DQ,-DP molecules under the given basal conditions (Fig. 1G). Costaining of each APM
component with the Schwann cell marker S100 confirmed their expression specifically in
Schwann cells (Fig. 1, right column of panels).
Cultured Human Schwann Cells Increase Expression of Delta (Y), TAP2, and HLA Class I
and HLA-DR,-DQ,-DP Under Inflammatory Conditions
We analyzed expression of APM components in primary human Schwann cell cultures
under basal conditions by flow cytometry as a different method and data were overall
consistent between both methods (summarized in Table 3). We next investigated regulated
expression following cytokine stimulation for 48 h by flow cytometry analysis. Following
IFN- stimulation (500 U mL1) we found increased expression of proteasome subunit delta
(Y) (Fig. 2A) and the antigenic peptide transport molecule TAP2 (Fig. 2B). Schwann cells
also increased expression of HLA Class I (Fig. 2C) and HLA-DR,-DQ,-DP molecules (Fig.
2D) after IFN- stimulation. Simultaneous staining with propidium iodide (PI) after IFN-
stimulation confirmed near total viability of the Schwann cell cultures used for subsequent
intracellular staining and flow cytometry analyses (Supp. Info. Fig. 2). Expression of the
other APM components was not altered to a relevant extent by IFN-. Stimulation with IL-2
and IL-4 had no detectable effects on the expression of any of the APM components
(summarized in Table 3).
Human Schwann Cells Express Main Components of the APM Under Basal Conditions In
Vivo
To correlate our in vitro results we analyzed APM component expression (Table 1) in
human Schwann cells in peripheral nerve sections from noninflammatory control patients
(Table 2). We used both a fluorescence-based (Fig. 3, second row) and a DAB-based (Fig. 3,
fourth row) staining approach. Fluorescent colocalization with the Schwann cell marker
S100 (Fig. 3, third row) confirmed Schwann cell derived staining. In this setting, human
Schwann cells expressed both the MB1 (X) (data not shown) and delta (Y) proteasome
subunits (Fig. 3A). The immunoproteasome subunit LMP2 was expressed in Schwann cells
(Fig. 3B), while LMP7 and LMP10 did not show immunoreactivity (data not shown). TAP2
(Fig. 3C) and Tapasin (Fig. 3D) exhibited prominent signal, while TAP1 did not stain
positive (data not shown). Schwann cells also expressed calnexin (Fig. 3E) and calreticulin

(Fig. 3F). Furthermore, human Schwann cells expressed HLA Class I molecules (Fig. 3G).
Staining against HLA-DR,-DQ,-DP returned weak signal in peripheral nerve sections from
noninflammatory control patients (Fig. 3H). Background staining from isotype control
staining was low to absent (Fig. 3I).
Peripheral Nerve Sections of GBS Patients

We obtained peripheral nerve sections from acute demyelinating GBS patients (Table 2).
The presence of T lymphocytes and macrophages in each individual nerve section had been
previously confirmed using immunohistochemistry (Hu et al., 2003; Kiefer et al., 1998). All
tissue sections from GBS patients (n = 5) showed active disease with foci of inflammatory
cells as determined by staining with different macrophage and T cell specific antibodies
(Table 1). Increased numbers of CD68 positive cells, indicative of macrophages, could
primarily be detected in the endoneurium and in perivascular cuffs in the epineurium. CD3
positive T lymphocytes could be localized to perivascular infiltrates in the epineurium, while
they were diffusely distributed within the endoneurium. The densities of endo- and epineural
T cells and different macrophage subsets in the individual nerve sections are given in Table
3. In subsequent correlation analyses we found a significant positive correlation (r = 0.8893,
P = 0.0435) between the densities of 25F9 positive late macrophages and staining intensity
of proteasome subunit delta (Y) in the respective sections. All other correlations between
APM staining intensity and the extent of inflammatory infiltration were analyzed and found
to be not significant (data not shown). Interestingly, individual T lymphocytes could be
localized in close proximity of Schwann cells (Supp. Info. Fig. 1D-I). In the
noninflammatory control group no inflammatory infiltrates were observed and
immunoreactivity against CD3 and CD68 was sparse. Control immunohistochemical
analyses after omission of the primary antibody showed only background staining for each
antibody used in the present study.
Schwann Cells in Peripheral Nerve Sections of GBS Patients Increase Expression of the
APM
Given our in vitro findings we asked whether an inflammatory setting would influence
expression of the APM components in Schwann cells also in vivo. For this purpose we
performed a fluorescent costaining of the Schwann cell marker S100 and all APM
components and visually rated Schwann cell derived staining intensity. Proteasome subunit
delta (Y) staining was more prominent in nerve sections from GBS patients (Fig. 4B)
compared with the control patients (Fig. 4A). Staining against the antigen peptide
transporter TAP2 appeared more obvious in nerve sections from GBS (Fig. 4D) than from
control patients (Fig. 4C). Also, HLA Class I staining was stronger in GBS patient-derived
(Fig. 4F) than in control patient-derived (Fig. 4E) peripheral nerve tissue. While staining
against HLA-DR,-DQ,-DP was faint to absent in Schwann cells from control patients (Fig.
4G), it was obvious in Schwann cells from GBS patients (Fig. 4H). Isotype control staining
was hardly detectable (Fig. 4I). Other APM components were expressed (Table 3), but
remained unaltered when comparing GBS to control samples. This visual rating suggested
that Schwann cells upregulate the expression of proteasome subunit delta (Y), antigen
peptide transported TAP2 and HLA Class I and II molecules in peripheral nerves from GBS
patients.
We sought to quantify our observation and assessed DAB-based immunohistochemical

staining intensity as a semi-quantitative measure of APM component expression. Sections
immediately adjacent to the ones used for DAB-based quantification were S100 stained to
exclude areas containing S100 negative cellsi.e. inflammatory cellsfrom quantification
of the adjacent DAB stained section. We further determined and compared Schwann cell

density in the sections and observed an insignificant trend towards lower Schwann cell
density in GBS nerves (data not shown).
Using this immunohistochemical quantification approach we found a significantly increased

staining intensity of proteasome subunit delta (Y) in inflamed peripheral nerves (Fig. 5A).
Further, expression of the peptide transporter TAP2 was significantly higher in GBS
compared with the control specimens (Fig. 5B). In contrast, analysis of the HLA peptide
loading molecule tapasin and the ER chaperone calreticulin yielded an only insignificant
trend towards elevated expression in GBS nerves (summarized in Table 3). Using this
quantification approach staining intensity of HLA Class I (Fig. 5C) and of HLA-DR,-DQ,-
DP (Fig. 5D) molecules was significantly higher in nerve sections from inflammatory
neuropathy patients when compared with noninflammatory controls.
Lysosomal Processing of Exogenous Ovalbumin by Cultured Human Schwann Cells

To analyze the ability of cultured human Schwann cells to incorporate and process
exogenous antigen we incubated the cells with DQ-Ovalbumina model antigen that only
returns fluorescent signal when proteolytically digested. Already within 1 h without any
proinflammatory stimulation Schwann cells began to develop green fluorescence, which
became more intensive within 3 h (Fig. 6A) and 6 h (Fig. 6B) of incubation. Schwann cells
without DQ-Ovalbumin exhibited no detectable autofluorescence and background signal
was low (data not shown). Freshly isolated human monocytes served as positive controls
and showed early and strong green fluorescence (Fig. 6C). Flow cytometry confirmed that
fluorescence from Schwann cells gradually increased following incubation with DQ-
Ovalbumin (Fig. 6D). Fluorescence from positive control monocytes (Fig. 6E) developed
faster and was generally stronger than fluorescence from Schwann cells (Fig. 6D). In a time
course analysis peak fluorescence intensity after 12 and 24 h of incubation, however, was
comparable between Schwann cells (red line) and monocytes (green line) (Fig. 6F). Staining
with propidium iodide (PI) confirmed that DQ-Ovalbumin did not affect the viability of the
Schwann cell cultures (Supp. Info. Fig. 2). The speed of exogenous antigen uptake is lower
in Schwann cells, but reaches a maximum comparable to unstimulated monocytes.
DISCUSSION
Reactivation of autoreactive neuritogenic T lymphocytes within the peripheral nerve is
considered to be one of the key steps in the immunopathogenesis of peripheral nerve
autoimmune disorders, such as GBS. Breakdown of myelin and Schwann cell components
could enhance processing and presentation of antigenic determinants by Schwann cells in
inflammatory neuropathies (Meyer zu Horste et al., 2008). Here, we show that human
Schwann cells express main components of the HLA antigen processing and presenting
machinery and increase their APM expression under inflammatory conditions in vitro and in
vivo. Our functional data further suggest that human Schwann cells are capable to uptake
and digest exogenous antigen at lower speed, but to a maximum comparable to unstimulated
monocytes. Our data thus support the hypothesis that antigen processing and presentation
may be an important feature of Schwann cells relevant for the pathogenesis of inflammatory
neuropathies. This does not contradict the concept that macrophages and dendritic cells
contribute to autoinflammation as professional APCs in the PNS (Kiefer et al., 2000; Muller
et al., 2006) and that humoral immunity co-mediates nerve damage in GBS (Yuki et al.,
2004). Our findings rather emphasize the importance of local cellular immune responses in
human inflammatory neuropathies.
Earlier studies have shown that Schwann cells express HLA molecules under certain
conditions. In vitro rodent (Ansselin and Pollard, 1990; Argall et al., 1991; Armati et al.,
1990; Duan et al., 2007; Lilje and Armati, 1997; Samuel et al., 1987) and human (Armati et

al., 1990; Spierings et al., 2001) Schwann cells constitutively express MHC Class I
molecules and can express MHC Class II molecules under proinflammatory conditions. Cell
surface levels of MHC Class I molecules can be elevated and expression of MHC Class II
molecules can be induced by proinflammatory cytokines (Ansselin and Pollard, 1990;

Bergsteinsdottir et al., 1992; Gold et al., 1995; Kingston et al., 1989; Lisak and Bealmear,
1992; Samuel et al., 1987; Tsai et al., 1991; Tsuyuki et al., 1998). HLA complexes have
been visualized in human peripheral nerve sections (Mancardi et al., 1988; Mitchell et al.,
1991; Pollard et al., 1987; Wanschitz et al., 2003), although data regarding expression of
HLA molecules by human Schwann cells in inflammatory conditions are sparse and
controversial (Schmidt et al., 1990). Coculture experiments demonstrated that rodent
Schwann cells can present endogenous and exogenous antigen to lymphocytes (Argall et al.,
1992a,b; Duan et al., 2007; Ford et al., 1993; Kingston et al., 1989; Lilje, 2002; Wekerle et
al., 1986), thereby providing evidence for a functional relevance of Schwann cell-derived
MHC expression. In human Schwann cells, presentation of exogenous mycobacterial
leprosy antigens has been observed (Spierings et al., 2001). However, whether human
Schwann cells regulate expression of HLA molecules under autoinflammatory conditions in
vivo remained controversial (Mitchell et al., 1991). Concerning the expression and
regulation of HLA molecules by Schwann cells, our study is in good principle agreement
with these previous findings. We demonstrate for the first time, however, expression and
regulation of the pathway of intracellular antigen processing in GBS patient derived material
and antigen uptake and digestion specifically in human Schwann cells.
We applied an immunohistochemical approach to quantify expression in vivo. This method

allows the quantification of embedded human peripheral nerve tissue which is more easily
available than RNA or protein preparations of unfixed nerve sections from human patients.
Initially, we visually assessed fluorescent signal intensity (see Fig. 4), which is clearly a
crude measure and susceptible to different types of bias. In an attempt to give our visual
impression a quantitative basis, we measured relative darkness of DAB-based staining; a
method which has been previously established (Matkowskyj et al., 2000, 2003). Naturally,
using this single stain approach one cannot fully exclude that staining intensity derives from
cell types other than Schwann cells. We therefore analyzed paraffin sections immediately
adjacent (5 m) to the individual ones used for DAB quantification (see Fig. 5) for the
presence of cells other than Schwann cells and excluded such areas from expression analysis
on the single stained sections. All staining and analyses were performed in parallel and in a
blinded fashion keeping intersection variability as low as possible. Concluding, we consider
our quantification method appropriate in our setting despite all the given limitations of
quantifying immunohistochemistry.
CD8+ cytotoxic T lymphocytes interact with HLA Class I molecules. Increased expression
of HLA Class I and parts of the corresponding antigen processing pathway in Schwann cells
in an inflammatory setting fits into the concept that CD8+ T cells significantly participate in
the pathogenesis of inflammatory neuropathies and corresponding animal models (Meyer zu
Horste et al., 2007). Indeed, both CD8+ cytotoxic T cells and CD4+ helper T cells have been
demonstrated in inflammatory infiltrates in GBS and CIDP (Cornblath et al., 1990; Kiefer et
al., 1998; Pollard et al., 1987; Schmidt et al., 1996). It is surprising, that in our analyses
Schwann cells expressed low amounts of HLA Class II molecules already under basal
conditions. This may reflect activation of Schwann cells in noninflammatory neuropathy
control patients. Comparison with the respective isotype controls (Figs. 3I and 4I) and the
previously demonstrated specificity of the antibodies argue for the adequacy of our staining
(Mehling et al., 2007; Ogino et al., 2003).
Interestingly, evidence from the literature indicates that in chronic inflammatory

neuropathies Schwann cells also express costimulatory molecules including B7-1, B7-2

(Murata and Dalakas, 2000) and CD58 (Van Rhijn et al., 2000). In an experimental leprosy
model cultured human Schwann cells expressed ICAM-1 and B7-1 (CD80) (Spierings et al.,
2001). Possible costimulators are thus available on Schwann cells, which argues against the
possible interpretation of our data that HLA molecules on Schwann cells induce anergy and
self-tolerance rather than propagating autoinflammation.
In summary, our study demonstrates the capability of human Schwann cells to process and
present antigens in vitro and in vivo and in a regulated fashion. This establishes Schwann
cells as possible facultative local antigen presenting cells and supports the hypothesis that
antigen processing and presentation in human Schwann cells is relevant in disabling
inflammatory neuropathies.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
The authors thank Olivia Kiehl, Bianca Wolff, and Tatjana Males for excellent technical assistance.
Grant sponsors: Forschungskommission of the Heinrich-Heine-University; Fritz-Thyssen-Stiftung; Deutsche

Forschungsgemeinschaft.
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Fig. 1.
Cultured human Schwann cells express antigen processing and presenting molecules.
Primary human Schwann cells under basal conditions were costained for S100 (left panels)
and APM components (middle panels); overlays are shown in the right panels.
Representative staining of duplicate analyses are shown and scale bars represent 5 m.
Fluorescent immunocytochemistry demonstrated expression of proteasome subunit delta (Y)
(A), immunoproteasome subunit LMP2 (B), and transporter associated with antigen
presentation TAP2 (C). Tapasin (D) and calnexin (E) were also expressed. Staining against
HLA Class I molecules was prominent (F), while staining against HLA-DR,-DQ,-DP
returned weak signal (G). Background signal in isotype control staining was low (H). [Color
figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 2.
Interferon- increases expression of proteasome subunit delta (Y), TAP2, HLA Class I, and
HLA Class II molecules in cultured human Schwann cells. Human Schwann cells were
stimulated with interferon (IFN)- for 48 h and analyzed for APM expression by flow
cytometry. Representative histograms of triplicate analyses depict overlays of isotype
control (open dashed line), unstimulated Schwann cells (gray filled line) and IFN- (500 U
mL1) stimulated Schwann cells (open continous line). Expression of proteasome subunit
delta (Y) was higher in IFN- stimulated (mean fluorescence intensity (MFI) 1444)
compared with the unstimulated Schwann cells (MFI 570) and isotype control stained cells
(MFI 19). Fluorescence returned from staining against TAP2 was higher in stimulated (MFI
843) than in unstimulated Schwann cells (MFI 252) (B). IFN- induced the expression of
HLA Class I (MFI 3400 vs. MFI 432) (C) and HLA-DR,-DQ,-DP molecules (MFI 1057 vs.
230) (D).

Fig. 3.
Human Schwann cells express main components of the antigen processing and presenting
machinery in vivo. Sural nerve sections from noninflammatory control patients were stained
for APM components using TRITC fluorescent (second column of panels) or DAB staining
(last column of panels). Costaining with S100 (first column of panels) confirmed Schwann
cell specific expression. Scale bars represent 5 m unless indicated otherwise.
Representative staining of five analyzed biopsy specimens are shown.
Immunohistochemistry documented expression of the proteasome subunit delta (Y) (A) and
LMP2 (B) by Schwann cells in vivo. Schwann cells expressed TAP2 (C) and Tapasin (D).
Staining against Calnexin (E) and Calreticulin (F) could be detected. HLA Class I (G) and
to a lower extentHLA Class II (H) molecules were expressed by human Schwann cells.
Isotype control staining showed only weak background staining (I).

Fig. 4.
Increased immunoreactivity of APM components in Schwann cells of GBS patients.
Fluorescent costaining of APM components and S100 were visually compared between sural
nerve biopsies from inflammatory neuropathy and control patients. Staining of proteasome
subunit delta (Y) in Schwann cells was stronger in GBS (B) compared with the control
samples (A). Also, TAP2 staining was more obvious in Schwann cells from GBS (D) than
from control patients (C). HLA Class I staining was stronger in GBS patient-derived (F)
than in control patient-derived (E) Schwann cells. While staining against HLA-DR,-DQ,-DP
was faint to absent in Schwann cells from control patients (G) it was obvious in Schwann
cells from GBS patients (H). Other APM components were expressed, but remained
unaltered when comparing GBS to control samples. [Color figure can be viewed in the
online issue, which is available at www.interscience.wiley.com.]

Fig. 5.
Peripheral nerves of GBS patients increase expression of proteasome subunit delta (Y),
TAP2, and HLA Class I and II molecules. Peripheral nerve sections from noninflammatory
control patients (n = 5) and from GBS patients (n = 5) were stained to semi-quantitatively
measure regulated expression using a DAB-based approach. Staining intensities of
proteasome subunit delta (Y) (A), TAP2 (B) and of HLA Class I (C) and HLA-DR,-DQ,-DP
(D) molecules appeared increased in nerve biopsies from GBS patients by visual assessment
(left columns) and were significantly increased when staining intensity was quantified as
relative darkness ranging from black (255) to white (0) in five randomly chosen
microscopic fields per section (right column). Scale bars represent 10 m. [Color figure can
be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 6.
Human Schwann cells process exogenous model antigen DQ-Ovalbumin. Human Schwann
cell cultures and unstimulated human monocytes were incubated with DQ-Ovalbumin which
returns fluorescent signal upon proteolytical digestion and fluorescence was assessed
visually (AC) and by flow cytometry analysis (DF). Within 1 h of incubation Schwann
cells began to exhibit green fluorescence which further increased within 3 (A) and 6 h (B).
Monocytes returned prominent fluorescent signal (C). Flow cytometry confirmed that
fluorescence from Schwann cells gradually increased (D). Fluorescence from positive
control monocytes (E) developed faster and was generally stronger than fluorescence from
Schwann cells. Peak fluorescence intensity was reached after 12 h of incubation and was
comparable between Schwann cells (red line) and monocytes (green line) (F). Scale bars
represent 5 m. One representative out of four comparable experiments is shown (AF) and
average values standard deviation of triplicate wells are depicted (F). [Color figure can be
viewed in the online issue, which is available at www.interscience.wiley.com.]

TABLE 1
Antibodies Used in the Current Study Sorted by Application
Antibody Recognizing Raised in Dilution Origin or reference

APM panel
SJJ3 Proteasome MB1 (X) Mouse 1:50 (Bandoh et al., 2005)
SY-5 Proteasome delta (Y) Mouse 1:50 (Bandoh et al., 2005)
NB1 Proteasome Z Mouse 1:25 (Bandoh et al., 2005)
SY-1 LMP2 Mouse 1:50 (Bandoh et al., 2005)
HB2 LMP7 Mouse 1:50 (Bandoh et al., 2005)
TO-6 LMP10 Mouse 1:50 (Bandoh et al., 2005)
NOB1 TAP1 Mouse 1:50 (Wang et al., 2005)
NOB2 TAP2 Mouse 1:50 (Wang et al., 2005)
TO-3 Tapasin Mouse 1:400 (Ogino et al., 2003)
TO-11 Calreticulin Mouse 1:100 (Ogino et al., 2003)
TO-5 Calnexin Mouse 1:50 (Ogino et al., 2003)
HC-10 HLA class I (B, C) heavy Mouse 1:1,000 (Stam et al., 1986)
LGII-612.14 HLA-DR,-DQ,-DP Mouse 1:50 (Temponi et al., 1993)
Schwann cell markers

S100 S100 Rabbit 1:100 (DAKO, Hamburg, Germany)
GFAP GFAP Rabbit 1:100 (DAKO, Hamburg, Germany)
p75 p75-neurotrophin receptor Rabbit 1:200 (Alomone Labs, Jerusalem, Israel)
Secondary antibodies and isotype control
Anti-mouse Mouse IgGTRITC Rat 1:100500 (Invitrogen, Karlsruhe, Germany)
Anti-rabbit Rabbit IgGAlexa fluor 488 Goat 1:100500 (Invitrogen, Karlsruhe, Germany)
Anti-rabbit Rabbit IgGAlexa fluor 594 Goat 1:100500 (Invitrogen, Karlsruhe, Germany)
Anti-rat Rat IgGAlexa fluor 488 Goat 1:500 (Invitrogen, Karlsruhe, Germany)
Anti-mouse Mouse IgGbiotinylated Rat 1:100500 (DAKO, Hamburg, Germany)
Mouse IgG1 kappa Isotype control Mouse 1:50 (eBiosciences, San Diego, CA)
Markers of inflammatory infiltrates
MRP14 Early activated macrophages Mouse 1:400 (Odink et al., 1987)
27E10 Early activated macrophages Mouse 1:100 (Zwadlo et al., 1986)
25F9 Macrophages late during inflammation Mouse 1:50 (Zwadlo et al., 1986)
MRP8 Macrophages late during inflammation Mouse 1:50 (Odink et al., 1987)
Anti-CD3 T cells Rat 1:200 (Nova Nordisk, Hamburg, Germany)

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
TABLE 2
Clinical, Electrophysiological, and Histopathological Features of Patients Analyzed in the Study
Disease Disease Amp tibial dmL tib NCV tib Amp sur NCV sur T epi T endo MRP14 27E10 25F9 MRP8
Patient Diagnosis duration severity nerve (mV) n (ms) n (m/s) n (mV) n (m/s) (#/mm2) (#/mm2) (#/mm2) (#/mm2) (#/mm2) (#/mm2)
HORSTE et al.
GBS #1 GBS 18 d severe 0.45 8.8 28 10.9 58 5.82 2.02 16.67 53.7 27.78 11.11
GBS #2 GBS 6d severe 12.6 9.2 42 16.9 39 56.5 38.46 0 9.09 18.18 9.09
GBS #3 GBS 13 d very severe 1.95 7.4 43 5.35 45 5.29 6.75 28.57 97.62 59.52 23.81
GBS #4 GBS 8d severe 1.26 7.4 45 17.2 40 4.91 0 10.34 0 13.79 10.34
GBS #5 GBS 3d moderate 10.1 6.4 43 16.9 52 5.23 0 0 0.76 2.27 0.76
Co #1 HNPP n.a. n.a. n.a. n.a. n.a. n.a. n.a. 1.09 2.17 0 0 1.39 1.39
Co #2 Toxic NP n.a. n.a. n.a. n.a. n.a. n.a. n.a. 19.44 2.63 0 1.16 1.16 2.33
Co #3 None n.a. n.a. n.a. n.a. n.a. n.a. n.a. 0 0 0 0 3.57 0
Co #4 HNPP n.a. n.a. n.a. n.a. n.a. n.a. n.a. 3.53 3.51 18.52 0 4.94 3.7
Co #5 Radiculitis n.a. n.a. n.a. n.a. n.a. n.a. n.a. 2.15 0 5.48 0 0 0
Disease duration in days after onset at the time of sural nerve biopsy is given in the third column. The last six columns give densities of T cells and macrophage populations in sural nerve biopsy cross sections after staining with the indicated markers as measured in cells per
mm2. MRP14 (Odink et al., 1987) and 27E10 (Zwadlo et al., 1986) stain early activated macrophages. 25F9 (Zwadlo et al., 1986) and MRP8 (Odink et al., 1987) stain macrophages late during inflammation. Co, control; GBS, Guillain-Barr-syndrome; HNPP, hereditary
neuropathy with liability to pressure palsies; NP, neuropathy; d, days; Amp, amplitude; dmL, distal motor latency; tib n, tibial nerve; sur n, sural nerve; NCV, nerve conduction velocity; T endo, endoneural CD3+ T cell density; T epi, epineural CD3+ T cell density; n.a. not
available.

Page 20
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
TABLE 3
Summary of Experimental Results
ICC SC in vitro FC SC in vitro FC SC IFN--fold change FC SC IL-2/IL-4 IHC SC in vivo IHC SC GBS-fold change
Depicted in figure 1 2 2 3 4
HORSTE et al.
Constitutive Proteasome
Delta (Y) +++ +++ 2.53 (Down) +++ 1.23
MB1 (X) (+) + 2.24 = + =
Z + = = n.a.
Immunoproteasome
LMP2 + + 2.04 = + =
LMP7 n.a. n.a. n.a.
LMP10 1.93 n.a. =
Peptide transfer to ER
TAP1 (+) n.a. n.a. n.a.
TAP2 ++ + 3.35 = + 1.29
Tapasin (+) + 3.29 = ++ =
ER chaperones
Calnexin +++ ++ = = +++ =
Calreticulin ++ 2.13 = ++ =
HLA-complexes
HLA Class I heavy ++ ++ 7.05 = ++ 1.29
HLA-DR,-DQ,-DP + (+) 4.60 = + 1.40

Human Schwann cells constitutively express main components of the APM in vitro. Expression is increased by proinflammatory IFN- and unaltered following stimulation with IL-2 or IL-4. Constitutive
APM component expression in vivo is increased in GBS samples. Column 4 describes regulated expression as relative fold change of stimulated versus non-stimulated cells. Column 7 describes regulated
expression as relative fold change of GBS versus non-inflammatory control patient samples. ICC, Immunocytochemistry; IHC, Immunohistochemistry; GBS, Guillain-Barr syndrome; SC, Schwann cell;
FC, flow cytometry; ER, endoplasmatic reticulum; IL-2, interleukin-2 stimulation; IL-4, interleukin-4 stimulation; IFN-, interferon- stimulation; n.a., not available; +++/++/+//=, high/medium/low/
no/not significantly regulated expression.
Page 21

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Glia. 2010 January 1; 58(1): 8092. doi:10.1002/glia.20903.

Expression of Antigen Processing and Presenting Molecules by

Heinrich-Heine-University, Dsseldorf, Germany

2009 Wiley-Liss, Inc.

Putative autoreactive T lymphocytes are under continuous control to ensure self-tolerance

of GBS, such cross-reactivity of anti-microbial antibodies towards endogenous nerve-

targets for modulating clinical disease manifestations in inflammatory neuropathies in the

Glia. Author manuscript; available in PMC 2012 October 25.

Processing of DQ-Ovalbumin by Cultured Human Schwann Cells

Flow Cytometry of Schwann Cells

Human Nerve Biopsies

Glia. Author manuscript; available in PMC 2012 October 25.

Analysis of Staining Intensity

Data Acquisition and Analysis

Glia. Author manuscript; available in PMC 2012 October 25.

Presenting Machinery at Basal Conditions In Vitro

(summarized in Table 3).

Glia. Author manuscript; available in PMC 2012 October 25.

staining was low to absent (Fig. 3I).

Peripheral Nerve Sections of GBS Patients

We sought to quantify our observation and assessed DAB-based immunohistochemical

Glia. Author manuscript; available in PMC 2012 October 25.

Using this immunohistochemical quantification approach we found a significantly increased

Lysosomal Processing of Exogenous Ovalbumin by Cultured Human Schwann Cells

Glia. Author manuscript; available in PMC 2012 October 25.

molecules can be induced by proinflammatory cytokines (Ansselin and Pollard, 1990;

We applied an immunohistochemical approach to quantify expression in vivo. This method

Interestingly, evidence from the literature indicates that in chronic inflammatory

Glia. Author manuscript; available in PMC 2012 October 25.

Grant sponsors: Forschungskommission of the Heinrich-Heine-University; Fritz-Thyssen-Stiftung; Deutsche

MHC molecules. Muscle Nerve. 1990; 13:106116. [PubMed: 2107399]

Glia. Author manuscript; available in PMC 2012 October 25.

Glia. Author manuscript; available in PMC 2012 October 25.

Mehling M, Simon P, Mittelbronn M, Meyermann R, Ferrone S, Weller M, Wiendl H. WHO grade

Two calcium-binding proteins in infiltrate macrophages of rheumatoid arthritis. Nature. 1987;

biopsies in Guillain-Barre syndrome and chronic inflammatory demyelinating neuropathy. Muscle

Glia. Author manuscript; available in PMC 2012 October 25.

Tsuyuki Y, Fujimaki H, Hikawa N, Fujita K, Nagata T, Minami M. IFN-gamma induces coordinate

12):25912625. [PubMed: 12429589]

Glia. Author manuscript; available in PMC 2012 October 25.

Glia. Author manuscript; available in PMC 2012 October 25.

Glia. Author manuscript; available in PMC 2012 October 25.

Glia. Author manuscript; available in PMC 2012 October 25.

Glia. Author manuscript; available in PMC 2012 October 25.

Glia. Author manuscript; available in PMC 2012 October 25.

Glia. Author manuscript; available in PMC 2012 October 25.

Antibody Recognizing Raised in Dilution Origin or reference

Schwann cell markers

Glia. Author manuscript; available in PMC 2012 October 25.

Glia. Author manuscript; available in PMC 2012 October 25.

Glia. Author manuscript; available in PMC 2012 October 25.

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