Patterns of protein expression in infectious meningitis: A cerebrospinal fluid protein array analysis

www.elsevier.com/locate/jneuroim

Journal of Neuroimmunolog

Patterns of protein expression in infectious meningitis:

A cerebrospinal fluid protein array analysis

Stefan Kastenbauer*, Barbara Angele, Bernd Sporer, Hans-Walter Pfister, Uwe Koedel

Department of Neurology, Klinikum Grosshadern, Ludwig-Maximilians-University, Marchioninistr. 15, 81377 Munich, Germany

Received 16 November 2004; accepted 21 March 2005

Abstract

Seventy-nine cytokines, chemokines, and growth factors were measured by protein array analysis in the cerebrospinal fluid of

patients with meningitis and controls. Several factors were found to be regulated, which have not been studied in the CNS before, e.g.,

macrophage inflammatory protein-1delta (CCL15) and neutrophil-activating peptide-2 (CXCL7). In pneumococcal meningitis, other new

observations were an increase of macrophage migration inhibitory factor, monocyte chemoattractant protein-2 (CCL8), pulmonary and

activation-regulated chemokine (CCL18), and macrophage inflammatory protein-3alpha (CCL20), and a sustained upregulation of

several growth factors. In viral meningitis, new findings were an elevation of CCL8, thrombopoietin, and vascular endothelial growth

factor.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Meningitis; Protein array; Cytokine; Chemokine; Growth factor

1. Introduction

The new technology of protein array analysis allows for

the simultaneous quantification of a large number of

proteins in one sample. We have used this method in order

to describe patterns of protein expression in the cerebrospi-

nal fluid (CSF) of patients with viral and pneumococcal

meningitis. We chose an array of 79, some of them only

recently identified, cytokines, chemokines, and growth

factors, because our understanding of these mediators of

leukocyte trafficking, inflammation and possibly meningi-

tis-associated neuronal injury is incomplete (Nau and Bruck,

2002; Koedel et al., 2002; Scheld et al., 2002; Ransohoff,

2002; Tauber and Moser, 1999; Lahrtz et al., 1998). The

main intention of this study was to identify new mediators,

which may be involved in the pathophysiology of bacterial

and viral meningitis.

0165-5728/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.jneuroim.2005.03.009

* Corresponding author. Tel.: +49 89 7095 0; fax: +49 89 7095 6673.

E-mail address: [email protected] (S. Kastenbauer).

2. Materials and methods

2.1. Patients

Lumbar punctures were performed for diagnostic pur-

poses after informed consent. After centrifugation, CSF

samples were stored at �30 -C until analysis. The

following patient groups were studied: pneumococcal

meningitis (acute stage), pneumococcal meningitis (fol-

low-up), viral meningitis, and controls. Control patients

(n =10) suffered from non-inflammatory diseases of the

nervous system: after extensive diagnostic work-up, their

diagnoses (n) were migraine (8), cervical disc herniation

(1), and epileptic seizure secondary to cerebral micro-

angiopathy (1). Their CSF findings were normal

(meanTS.D.: 2T1 leukocytes/Al, 33T17 mg/dl protein).

Patients with pneumococcal meningitis (n =10) had typical

signs and symptoms of meningitis (fever, headache,

meningism), a neutrophil CSF pleocytosis (2568T1948leukocytes/Al), evidence of severe blood–CSF barrier

disruption (424T245 mg/dl protein), and a positive CSF

y 164 (2005) 134 – 139

S. Kastenbauer et al. / Journal of Neuroimmunology 164 (2005) 134–139 135

or blood culture for Streptococcus pneumoniae. From all

patients with pneumococcal meningitis, follow-up CSF

samples were available (167 T153 leukocytes/Al, 141T60mg/dl protein). They were obtained after a median of 5 days

(range 3–11) after the diagnostic lumbar puncture and

initiation of antibiotic treatment. Patients with viral menin-

gitis (n =10) had typical signs and symptoms of meningitis,

a lymphomonocytic CSF pleocytosis (337T342 leukocytes/

Al), evidence of mild blood–CSF barrier disruption (79T29mg/dl protein), normal blood leukocyte counts (<11 G/l),

and normal serum C-reactive protein levels (<0.5 mg/dl).

Sex distribution and age were not statistically different

between diagnostic groups.

2.2. Protein array

For each diagnostic group, the CSF samples were

pooled: from every patient, a CSF volume containing 50

Ag of protein was used, resulting in a total of 500 Ag protein

per group. The approach based on total protein instead of

volume was chosen, because the protein array does not

allow for the quantification of proteins by comparison with

a standard curve (which is impossible due to the multitude

of proteins). Therefore, the results had to be normalized by

Fig. 1. Cerebrospinal fluid protein array analysis of 79 cytokines, chemokines, and

controls (patients with non-inflammatory diseases), patients with pneumococcal

meningitis (for abbreviations, see Table 1).

comparison with the positive controls (see below), which

required the samples to contain equal amounts of total

protein. The RayBioi Human Cytokine Array V (http://

www.raybiotech.com) was used which detects 79 human

cytokines, chemokines, and growth factors. The array

membrane contains dots of antigen-specific immobilized

antibodies, arranged in 11 columns and 8 rows (Fig. 1). Six

dots are coated with biotin-conjugated IgG (positive

controls) and three are uncoated (negative controls). The

protein array was performed according to the manufacturer’s

instructions. In brief, the antigen-specific immunoreactivity

is detected with biotin-conjugated soluble antibodies and

horseradish peroxidase-conjugated streptavidin. Densitom-

etry of chemiluminescence-exposed X-ray films was used

for quantification. In order to normalize the results, the

optical densities of each dot were then expressed as

percentage of the average optical densities of the 6 positive

controls contained on each array membrane. Because the

protein array detects only relative expression levels and not

absolute values, there is no defined lower detection limit.

However, for the sake of specificity we limited the

sensitivity of the test by ignoring expression levels <5%

of the positive controls. Equal to or more than 2-fold

differences of the expression levels between diagnostic

growth factors in pooled samples (10 patients in every diagnostic group) of

meningitis on admission and during follow-up, and in patients with viral

http://www.raybiotech.com

Table 1

Alphabetical list of proteins under study

Abbreviation Name Systematic

name

(where

available)

Ang Angiopoietin

BDNF Brain-derived neurotrophic factor

BLC B-lymphocyte chemoattractant CXCL13

Ck h 8-1 Human beta-chemokine 8-1 CCL23

EGF Epidermal growth factor

ENA-78 Epithelial-cell-derived

neutrophil-activating protein

CXCL5

Eotaxin Eotaxin CCL11

Eotaxin-2 Eotaxin-2 CCL24

Eotaxin-3 Eotaxin-3 CCL26

FGF-4 Fibroblast growth factor-4




Flt-3 ligand Fms-like tyrosine kinase-3 ligand

Fractalkine Fractalkine CX3CL1

GCSF Granulocyte colony stimulating factor

GCP-2 Granulocyte chemotactic protein-2 CXCL6

GDNF Glial cell line-derived neurotrophic factor

GM-CSF Granulocyte macrophage-colony stimulating

factor

GRO Growth-related oncogene

GRO-a Growth-related oncogene-alpha CXCL1

HGF Hepatocyte growth factor

IFN-g Interferon-gamma

IGF-I Insulin like growth factor-I

IGFBP-1 Insulin-like growth factor binding protein-1



IBFBP-4 Insulin-like growth factor binding protein-4

IL-1a Interleukin-1 alpha

IL-1h Interleukin-1 beta

IL-2 Interleukin-2

IL-3 Interleukin-3

IL-4 Interleukin-4

IL-5 Interleukin-5

IL-6 Interleukin-6

IL-7 Interleukin-7

IL-8 Interleukin-8 CXCL8

IL-10 Interleukin-10





IP-10 Interferon-inducible cytokine-10 CXCL10

I-309 I-309 CCL1

Leptin Leptin

LIF Leukemia inhibitory factor

LIGHT Homologous to lymphotoxin, exhibits

inducible expression and competes with HSV

glycoprotein D for herpes virus entry mediator,

a receptor expressed on T cells

TNFSF14

MCP-1 Monocyte chemoattractant protein-1 CCL2




MCSF Macrophage colony stimulating factor

MDC Macrophage-derived chemokine CCL22

MIF Macrophage migration inhibitory factor

MIG Monokine induced by IFN-g CXCL9

Abbreviation Name Systematic

name

(where

available)

MIP-1h Macrophage inflammatory protein-1 beta CCL4

MIP-1y Macrophage inflammatory protein-1 delta CCL15

MIP-3a Macrophage inflammatory protein-3 alpha CCL20

NAP-2 Neutrophil activating peptide-2 CXCL7

NT-3 Neurotrophin-3

NT-4 Neurotrophin-4

OSM Oncostatin

OPG Osteoprotegerin

PARC Pulmonary and activation-regulated chemokine CCL18

PDGF-B Platelet-derived growth factor-B

PIGF Placental growth factor

RANTES Regulated on activation normal T cell

expressed and secreted

CCL5

SCF Stem cell factor

SDF-1 Chemokine stromal-derived factor-1 CXCL12

TARC Thymus and activation-regulated chemokine CCL17

TGF-h1 Transforming growth factor-beta1

TGF-h2 Transforming growth factor-beta2

TIMP-1 Tissue inhibitor of metalloproteinases-1

TIMP-2 Tissue inhibitor of metalloproteinases-2

TNF-a Tumor necrosis factor-alpha

TNF-h Tumor necrosis factor-beta

TPO Thrombopoietin

VEGF Vascular endothelial growth factor

Table 1 (continued)

S. Kastenbauer et al. / Journal of Neuroimmunology 164 (2005) 134–139136

groups were considered as significant. The proteins under

study are listed in Table 1.

3. Results

On admission, patients with pneumococcal meningitis

had �2-fold elevated CSF levels (compared with controls)

of the cytokines IL-1h, IL-6, IL-10, MIF, and TNF-a, of the

CXC chemokines ENA-78, GRO, IL-8, IP-10, and NAP-2,

of the CC chemokines MCP-1, MCP-2, and MIP-3a, and of

the growth factors HGF, IGFBP-1, and MCSF (see Fig. 1).

During recovery from pneumococcal meningitis, IL-1h,IL-10, MIF, TNF-a,ENA-78, MCP-2, MIP-3a,and MCSF

returned to control levels. However, IL-6, GRO, IL-8, NAP-

2, IP-10, HGF, and IGFBP-1 were still elevated. Interest-

ingly, the CC chemokines MIP-1y and PARC and the

growth factors IGFBP-4 and PIGF showed a marked

increase at follow-up (see Fig. 1).

Patients with viral meningitis had markedly increased

CSF levels of the cytokines IL-6 and IL-10, of the CXC

chemokines GRO, IL-8 and IP-10, of the CC chemokines

MCP-2 and MIP-1y, and of the growth factors NT-3, TPO,

and VEGF compared with controls (see Fig. 1).

The following proteins were reliably detectable (�5% of

the positive controls) in any diagnostic group but were not

markedly regulated during the diseases under study (not

�2-fold different between diagnostic groups): the cytokines

IFN-g, IL-1a, IL-2, IL-16, LIF, LIGHT, MIG, and TGF-h3,


the CXC chemokine SDF-1, the CC chemokines eotaxin-2

and MIP-1h, the CX3C chemokine fractalkine, and the

growth factors Ang, EGF, FGF-4, FGF-9, GDNF, GCSF,

IGFBP-2, IGFBP-3, NT-4, OSM, and SCF.

TIMP-2, the cytokine TGF-h2, and the growth factors

Flt-3 and OPG were �5% of the positive controls in control

patients and were not increased but decreased �2-fold in

any group of patients with meningitis.

Finally, the following proteins were ignored in the

present study because their expression levels were <5% of

the positive controls in all diagnostic groups: the cytokines

IL-3, IL-4, IL-5, IL-7, IL-12, IL-13, IL-15, TGF-h1, andTNF-h, the CXC chemokines GCP-2, GRO-a, and BLC,

the CC chemokines Ck h 8-1, eotaxin, eotaxin-3, I-309,

MCP-3, MCP-4, MDC, RANTES, and TARC, and the

growth factors FGF-6, FGF-7, GMCSF, IGF-I, PDGF-B,

and leptin.

4. Discussion

The intention of this study was to characterize patterns of

protein expression during infectious meningitis. The protein

array was not based on CSF volume, but on CSF total

protein in order to normalize the results by comparison with

the positive controls. Therefore, our results may disagree

from previously published volume-based tests (e.g., ELISA

studies) and our assay may have underestimated the increase

of some proteins in CSF. False positive results are, there-

fore, very unlikely, but false negative results (i.e. observa-

tion of a decrease or no change of a protein previously

reported to be increased by means of volume-based tests)

are possible. Therefore, negative results were ignored and

only positive results are discussed here.

First of all, the detection of the cytokine LIGHT, of the

CXC chemokine NAP-2, of the CC chemokines eotaxin-2,

MCP-2, MIP-1y, MIP-3a,and PARC, and of the growth

factors angiogenin, FGF-4, FGF-9, Flt-3 ligand, oncostatin,

PIGF, and SCF was an interesting finding, because we are

not aware of any previous publication reporting their

presence in human CSF. Some of these proteins have not

even been detected in human or animal brains or brain cell

cultures. For example, the cerebral expression of PARC has

been investigated only in one study, which failed to detect

its RNA in brain, while it was abundantly expressed in

monocytes and lung tissue (Guan et al., 1999). The

biological functions of PARC are largely unknown, but it

has been shown to exert chemotactic effects on monocytes,

lymphocytes, and immature dendritic cells (Vulcano et al.,

2003; Schraufstatter et al., 2004). To the best of our

knowledge, the expression of MIP-1y, eotaxin-2, and

NAP-2 has not been studied at all in human or animal

brains or brain cells. In vitro studies have shown that MIP-

1y can be induced in monocytes and is chemoattractive for

T-lymphocytes and monocytes (Coulin et al., 1997).

Eotaxin-2 is of major importance as an eosinophil chemo-

attractant in allergic diseases (Menzies-Gow et al., 2002). In

the lung, it was shown to be expressed by bronchial

epithelial cells, and to a lesser degree by endothelium and

monocytes (Ying et al., 1999). NAP-2 which is truncation

product of the platelet-derived connective tissue-activating

peptide III, is a strong chemoattractant and activator of

neutrophils (Schenk et al., 2002). The sources of these

proteins and their functions in the CNS remain elusive and

need to be studied further.

The increased levels of the cytokines IL-1h, IL-6, IL-10,and TNF-a, of the CXC chemokines ENA-78, GRO, and

IL-8, of the CC chemokines MCP-1 and IP-10, and of the

growth factors HGF and MCSF during pneumococcal

meningitis are confirmatory of previous studies of acute

bacterial meningitis (Zwijnenburg et al., 2003; Koedel et al.,

2002; Tauber and Moser, 1999; Lahrtz et al., 1998; Nayeri et

al., 2000; Pashenkov et al., 2002).

Moreover, we also made several new observations in our

sample of patients with pneumococcal meningitis during the

acute stage.

First, the increase of the CC chemokines MCP-2 and

MIP-3aand of the CXC chemokine NAP-2 has not been

reported before in bacterial meningitis; they might play a

role in leukocyte extravasation during meningitis, as in vitro

experiments and animal studies of other infectious diseases

have shown that these proteins play a role in leukocyte

migration (Uguccioni et al., 1995; Bennouna et al., 2003;

Doroshenko et al., 2002). MCP-2 is thought to play a role in

the pathogenesis of multiple sclerosis, because it has been

detected in leukocytes (in particular, in macrophages) and

astrocytes in MS lesions but not in normal control brains

(McManus et al., 1998). We are not aware of any study

investigating MIP-3a in the human nervous system, but it

has been shown to be upregulated in the central nervous

system of mice with experimental autoimmune encephalo-

myelitis and in mouse brain-derived astrocytes upon

stimulation with IL-1h or TNF-a (Ambrosini et al., 2003).

Second, the increased expression of MIF during bacterial

meningitis is also a new finding; MIF is an important

proinflammatory regulator of innate immunity (Calandra

and Roger, 2003). In the rat brain, MIF has been shown to

be expressed mostly in neurons and astrocytes; infection

with Borna disease virus lead to an upregulation of MIF in

astrocytes, tanocytes, ependyma and choroid plexus epithe-

lium (Bacher et al., 2002). Its role in meningitis remains to

be determined.

Third, in bacterial meningitis research, elevated CSF

levels of IGFBP1 (and of IGFBP-4 during recovery) have

not been reported before; these binding proteins might play

a beneficial role, because they facilitate the transportation of

the neurotrophic IGFs (Walter et al., 1999).

During recovery from pneumococcal meningitis, we

observed a sustained upregulation of factors with neuro-

trophic properties: IL-6, HGF, and IGFBP-1 remained

elevated and PIGF showed a slight and IGFBP-4 a marked

increase. This increase of neurotrophic factors might reflect

S. Kastenbauer et al. / Journal of Neuroimmunology 164 (2005) 134–139138

an effort to limit meningitis-associated tissue and, in

particular, neuronal injury. The sustained increase of IL-6

is particularly interesting, because–in addition to its neuro-

trophic properties (Van Wagoner and Benveniste, 1999)

which still remain to be investigated in bacterial meningi-

tis–it has been shown to limit the inflammatory response

and to contribute to blood–brain barrier disruption in

experimental pneumococcal meningitis (Paul et al., 2003).

The pathophysiological role of the other above-mentioned

trophic factors in bacterial meningitis still remains to be

determined. In other animals models, however, HGF was

shown to protect against ischemic brain damage and

neuronal injury (Shimamura et al., 2004) and overexpres-

sion of IGFBP-1 reduced reactive astrocytosis in brain

trauma (Ni et al., 1997). PIGF has been shown to contribute

to vascular permeability in different experimental conditions

(Luttun et al., 2002). Overexpression of PIGF in the

hippocampus had negative effects on neurogenesis and

inhibited learning in mice (Cao et al., 2004); its role in the

diseased nervous system, however, still remains to be

determined.

The late increase of PARC and MIP-1y during pneumo-

coccal meningitis demonstrates for the first time that these

novel CC chemokines (Wang et al., 1998; Hieshima et al.,

1997) are regulated during meningitis, underscoring that

their role in neuroinflammation deserves further attention.

In viral meningitis, many of our observations are in good

agreement with previous studies, e.g., the increase of the

cytokines IL-6 and IL-10, of the CXC chemokine IL-8, and

of the CC chemokine IP-10 (Tauber and Moser, 1999;

Lahrtz et al., 1998). Several of our results, however, are

new, namely the increase of the CC chemokines MCP-2 and

MIP-1y and of the growth factors NT-3, TPO, and VEGF.

Similar to bacterial meningitis, MCP-2 (Uguccioni et al.,

1995) and MIP-1y (Wang et al., 1998) might play a role in

leukocyte trafficking into the CNS during viral meningitis.

Constitutive expression of TPO has been detected in human

brain and cerebrospinal fluid (Dame et al., 2003) but we are

not aware of any study of TPO expression in the diseased

nervous system. This regulator of megakaryopoiesis and

platelet production contains a neurotrophic sequence and

may therefore play a role in neuronal biology (Dame et al.,

2003). Surprisingly, a recent publication showed a proa-

poptotic effect of thrombopoietin on neurons (Ehrenreich et

al., 2005). Its function during neuroinflammation, however,

has not been characterized yet. Our observation of increased

levels of VEGF, which can promote vascular permeability

and vasogenic brain edema (Paul et al., 2001), is in contrast

to a previous study, which showed no change during viral

meningitis (van der Flier et al., 2001). However, as in that

study all patients with viral meningitis and all controls were

below the detection limit of the ELISA, our positive result

might be attributed to a higher sensitivity of the protein

assay. The finding of increased NT-3 also disagrees from a

previous study which failed to show elevated levels of NT-3

in children with viral meningitis, again maybe due to a

higher sensitivity of our assay (Mizuno et al., 2000). In

experimental autoimmune encephalomyelitis, T and NK

cells infiltrating the spinal cord were shown to express NT-3

(Hammarberg et al., 2000) and this observation has been

interpreted as an effort to curb the neurodamaging con-

sequences of CNS inflammation. Thus, it can be speculated

that NT-3 might play a similar role in viral meningitis.

Taken together, in this first protein array analysis of CSF

cytokines, chemokines, and growth factors in infectious

meningitis we demonstrate distinct patterns of protein

expression in viral meningitis and in the acute stage and

during recovery from pneumococcal meningitis. Our study

can be a basis for further investigations, which should

characterize the pathophysiological importance of the newly

identified mediators.

Acknowledgement

This study was supported by grants from the Deutsche

Forschungsgemeinschaft (SFB 576/TP A5 to UK and PF

246/6-1 to HWP).

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Patterns of protein expression in infectious meningitis: A cerebrospinal fluid protein array analysis