Neopterin in neuroborreliosis 
ENHANCED INTRA THECAL NEOPTERIN 
PRODUCTION AND TRYPTOPHAN DEGRADATION 
IN PATIENTS WITH LYME NEUROBORRELIOSIS 
D. Fuchs, L. Dotevall, Th. Gasse, L. Hagberg, G. P. Tilz, G. Baier-Bitterlich, 
U. Demel, E. Schmutzhard and H. Wachter 
ABSTRACT 
Various immune system compartments respond to Borrelia burgdo,feri infection. TH2-type and THl-type 
immune responses seem to be important. It recently was found that surface proteins of Borrelia burgdo,feri 
induce the formation of interferon-y in vitro. 
Upon stimulation with interferon-y, monocytes/macrophages release large amounts of neopterin and 
degradation of tryptophan is induced in a variety of cells. In humans increased neopterin concentrations and 
tryptophan degradation in body fluids indirectly indicate the endogenous formation of interferon-y and THl-
type immune response. 
Using serum and cerebrospinal fluid from patients with Lyme neuroborreliosis and with late Lyme 
encephalopathy, we investigated neopterin concentrations by radioimmunoassay and tryptophan and kynurenine 
by HPLC. 
Increased neopterin and kynurenine concentrations were found in cerebrospinal fluid but not in the serum 
of patients with Lyme neuroborreliosis, whereas tryptophan was decreased in cerebrospinal fluid. Abnormalities 
were related to the activity of the disease, during antibiotic treatment concentrations of neopterin and 
kynurenine tended to normalize. In patients with late Lyme encephalopathy the deviations of the 
analytes'concentrations from normal were only marginal compared to patients with acute Lyme neuroborreliosis. 
Increased formation of neopterin and degradation of tryptophan suggest a pathogenetic role of intrathecally 
released interferon-y and activated monocytic cells in patients with Lyme neuroborreliosis. 
KEYWORDS 
Borrelia burgdo,feri infection, Lyme neuroborreliosis, neopterin, tryptophan, kynurenine, inte,feron-y 
INTRODUCTION 
Infections with common bacteria lead to a cascade 
of immunological reactions. The front line of immune 
reaction to bacteria includes the immunological 
processing of pathogens by monocytes, the exchange 
acta dermatovenerologica A.P A. Vol 5, 96, No 3-4 
of signals between monocytes and lymphocytes and 
the formation of antibodies by activated B lymphocytes. 
The cytokines primarily involved in this so-ca}led 
TH2-type immune response comprise mainly inter-
leukins-4, -5 and -10. The situation is even more 
complex concerning intracellular bacteria or viruses. 
137 
Neopterin in neuroborreliosis 
In this case the type of immune response is more 
on the side of the T-cell/macrophage axis and THl-
type cytokines, interferon-y and interleukin-2, play a 
more important role. In the end, certain antigens or 
toxins of preferentially Gram-negative bacteria are 
able to directly activate T-lymphocytes for cytokine 
production; this kind of immune reaction mainly 
involves THl-type cytokines such as interferon-y. 
There is a cross-regulation of THl-type and TH2-
type immune responses (1). Cytokines involved in 
THl- or TH2-type immune response down-regulate 
each other. 
Various immune system compartments respond to 
Bomlia burgdorferi (Bb) infection. TH2-type immune 
response is easily documented in patients by determi-
ning the antibody seroconversion after the acute 
infection (2). Apart from TH2-type immunity, THl-
type immune response especially seems to play a 
major role in the later course of infection when the 
primary immune response has been unable to clear 
the organism from the invading spirochetes. Moreover, 
it was discovered recently that Bb itself is able to 
induce formation of interferon-y (3,4) and it seems 
that interferon-y could play a role in the pathogenesis 
of Lyme neuroborreliosis (LNB). However, only 
little is known about the interferon-y system in Bb 
infection. This is partly due to the fact that direct 
measurement of interferon-y concentrations is com-
plicated because of the short biological half-life of 
this cytokine in vivo and the test systems generally 
applied also don't appear to be sensitive enough for 
studies of patients (5). 
Upon stimulation with interferon-y, human mono-
cytes/macrophages release large amounts of neopterin 
(6) and in parallel degradation of tryptophan is 
induced (Fig. 1) in a variety of cells (7 ,8). Increased 
neopterin concentrations and tryptophan degradation 
- estimated from tryptophan and kynurenine measure-
ments - in body fluids in patients indirectly indicate 
endogenous formation of interferon-y and THl-type 
immune response. Therefore, monitoring neopterin 
concentrations tumed out to be useful for follow-
up and for predicting the further clinical course in 
patients with infectious diseases including human 
immunodeficiency virus (HIV) infection, in 
autoimmune diseases and various types of malignancies 
(6,9). 
In this study we compared the results of three 
earlier investigations (10-12) in which we measured 
concentrations of neopterin, tryptophan and kynurenine 
in serum and cerebrospinal fluid (CSF) in patients 
with LNB and late Lyme encephalopathy. 
138 
METHODS 
Our studies comprised 18 patients (11 females, 7 
males, age: 28 - 70 years) with LNB and 10 patients 
(5 females, 5 males, age: 29 - 66 years) with late 
Lyme encephalopathy. The diagnosis LNB was defined 
as a) neurologic symptoms compatible with neuroborre-
liosis and a positive antibody test, and signs of 
intrathecal antibody production in CSF, b) neurologic 
signs and symptoms within three months after onset 
of erythema migrans, c) bilateral facial palsy in a 
child with CSF pleocytosis and no other known 
disease. In serum and CSF of patients neopterin 
concentrations were measured by radioimmunoassay 
(BRAHMS-Henning, Berlin, Germany). In addition, 
tryptophan and kynurenine concentrations were 
measured by high pressure liquid chromatography 
(HPLC) on reversed phase C18 material (Chromosorb, 
Merek, Darmstadt, Germany) as previously described 
(12). Compounds were quantified using fluorescence 
measurements in the case of tryptophan (285 nm 
excitation wavelength and 360 nm emission) and by 
detection of UV absorption in the case of kynurenine 
(360 nm wavelength; detection limit: < 0.1 µmol/1). 
The concentrations found in our patients were 
compared to previously established normal ranges 
obtained by implementing the same methodology. 
M onocyte / macrophage 
lnterferon-1 
/ ' Activation of Activation of 
indoleamine (2,3)-dioxygenase GTP-cyclohydrolase 1 
= =~= = = = = =!= = = = = = = = = =~= = = 
Tryptophan Kynurenine Neopterin 
Extracellular space 
Fig. l. Interferon-y activates guanosine triphosphate-
( GTP)-cyclohydrolase I in human monocytes/macrophages 
which leads to the fonnation of large amounts of 
neopterin. At the same tirne it induces indoleamine 
(2,3)-dioxygenase in a variety of cells which aegrades 
tryptophan via the kynurenine pathway, with kynurenine 
being one of its first products. 
acta dennatovenerologica A.P A. Vol 5, 96, No 3-4 
Neopterin in neuroborreliosis 
Interferon-, 
Tryptophan! C> Serotonin! Neopterinf 
I 
Kynureninic acid f I Oxidative stress f 
I 
Quinolinic acid f 
! 
Apoptosis f 
Fig. 2. Interferon-y mediated neopterin formation and 
tryptophan degradation could insult neuroendocrine tissue. 
On the one hand, degradation of tryptophan may 
reduce its availability for the biosynthesis of serotonin 
(left part); on the other hand, it may lead to the 
accumulation of degradation products, some of which 
like kynurenic acid and quinolinic acid, are patent 
neurotoxins inte,fering with the NMDA receptor. Increased 
concentrations of neopterin derivatives are able to 
enhance oxygen free-radical mediated processes such as 
apoptosis (right part). 
Matched serum and CSF specimens were collected, 
samples were kept frozen at -20°C until measurements 
were performed. 
Statistical evaluations of the data included the 
non-parametric Wilcoxon test for comparisons of 
grouped data and the Spearmen rank correlation 
analyses. 
RESULTS 
Increased concentrations of CSF neopterin (> 6 
nmol/1) were found in 16/18 patients with early or 
progressive LNB (mean ± S.D.: 31.8 ± 45.4 nmol/ 
l; controls: 3.2 ± 1.88) and neopterin levels correlated 
to the cell counts (rs = 0.775, p < 0.001) and 
protein content (rs = 0.812, p < 0.001) of the CSF 
(10,12). A subgroup of patients (67%) with LNB 
had detectable levels of CSF kynurenine (0.1 - 2.0 
µmol/1) and there was a positive correlation between 
the neopterin and kynurenine levels (rs = 0.791, 
. p< 0.001) (11,12). Preferentially those patients who 
presented with relatively low CSF neopterin con-
acta dennatovenerologica A.P A. Vol 5, 96, No 3-4 
centrations had undetectable levels of CSF kynurenine 
similar to controls. CSF tryptophan was below O.S 
µmol/1 in 3/18 patients, who showed the highest 
neopterin and kynurenine levels (11,12). The patient 
with the highest kynurenine (2.0 µmolil) and neopterin 
(183 nmol/1) concentrations in their CSF had 
undetectable levels of tryptophan ( < 0.2 nmol/1). 
Abnormalities were related to the activity of the 
disease and concentrations of neopterin tended to 
normalize during antibiotic treatment (10). There 
were only minor changes in the concentrations of 
analytes seen in the serum specimens (neopterin: 
7.8 ± 3.48 nmol/1) and only 3/18 patients had 
increased neopterin compared to healthy controls 
(10-12). Serum tryptophan tended to decrease and 
kynurenine to increase compared to controls (11,12). 
However, the absolute changes are much lower than 
those observed intrathecally. 
In patients with late Lyme encephalopathy the 
deviations of the analytes' concentrations in their 
CSF from normal were only marginal compared to 
patients with acute LNB (12). However, the changes 
took the same direction as was observed in patients 
with LNB: 1/9 patients had increased CSF neopterin 
and 2/9 patients had detectable levels of kynurenine 
in their CSF. It appeared that the serum concentrations 
of tryptophan decreased over the duration of the 
disease. In fact, the mean serum tryptophan was 
decreased (57.9 ± 8.8 µmolil) compared to the 
healthy controls (89.2 ± 15.6 µmolil) (12). 
DISCUSSION 
In our study we found increased levels of neopterin 
and kynurenine and decreased tryptophan concen-
trations in the CSF of patients with LNB. The 
correlation found between increased neopterin and 
tryptophan metabolic changes suggests that the 
decrease in tryptophan is due to degradation rather 
than a reduced dietary intake of this essential 
amina acid. The increased formation of neopterin 
and degradation of tryptophan was located inside 
the blood-brain-barrier as only minor changes could 
be observed in the serum of patients. In addition, 
these results were obtained almost exclusively in 
patients with LNB but not in patients with late 
Lyme encephalopathy. 
The intrathecal formation of neopterin and degra-
dation of tryptophan demonstrate the THl-type 
immune response taking place in patients (Fig. 1) 
and suggest a pathogenetic role of intrathecally 
released interferon-y and activated monocytic cells 
139 
Neopterin in neuroborreliosis 
in patients with LNB. It seems plausible that inter-
feron-y is formed in response to certain spirochetes' 
surface proteins which are known to stimulate the 
release of this cytokine in vitro (2,3). Alternatively, 
it could also result from a cell-mediated immune 
response to infected cells. It is also a fact that 
during other cerebral infections such as toxoplasmosis, 
measles or HIV infection (13-15) large amounts of 
neopterin are produced inside the blood-brain-barrier. 
Thus, an immune response is established locally and 
monocytic cells like microglia or astrocytes or 
monocytes/macrophages, which have permeated the 
blood-brain-barrier, are involved in the formation of 
increased neopterin. Interestingly a correlation was 
found between CSF cell counts and neopterin levels 
(10). However, in other situations like HIV infection, 
equally high neopterin levels have been found despite 
of low CSF cell counts (15). 
Enhanced formation of neopterin and tryptophan 
degradation could be involved in the pathogenesis 
of symptoms in LNB (Fig. 2). Neopterin derivatives 
have been described to potentially internet with 
reactive oxygen intermediates released by stimulated 
immunocompetent cells. In fact, neopterin was recently 
found to enhance the effects of hydrogen peroxide 
in vitro (16) and one role of neopterin derivatives 
was found to superinduce apoptosis triggered by 
tumor necrosis factor-a (17). Thus, loss of neuro-
endocrine tissue during cerebral Bb infection could 
be enhanced by the overwhelming and continuous 
production of neopterin derivatives within intrathecal 
THl-type immune response (18). Oxidative stress 
due to chronic activation of immunocompetent cells 
may be enhanced by the presence of large amounts 
of neopterin derivatives at local sites (19). 
Also tryptophan metabolic changes could play a 
role in precipitating symptoms in patients with LNB. 
Degradation of tryptophan leads to the accumulation 
of toxic products like kynurenic acid or quinolinic 
acid (18,20) inside the brain and, thus, may interfere 
with NMDA receptor mediated signaling pathways. 
Moreover, tryptophan is the precursor molecule of 
5-hydroxy-tryptamine (serotonin), an important neuro-
transmitter (Fig. 2). The availability of tryptophan is 
crucial for the biosynthesis of serotonin. Thus, 
decreased tryptophan may limit serotonin production 
which is especially important for neuroendocrine 
cells (8). 
It should be mentioned that abnormalities of 
neopterin and tryptophan concentrations have been 
observed almost exclusively in patients with LNB 
but not in those with late Lyme encephalopathy. 
This data favours the view that neurological sequelae 
are merely caused by the acute infectious process 
and there is no or only a very limited contribution 
from a chronic ongoing infection at the tirne of the 
sample collection 
This work was financially supported by the Austrian funds „Zur Forderung der wissenschaftlichen Forschung'; P 10776 
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AUTHORS' ADDRESSES 
Dietmar Fuchs, PhD, professor, Institute of Medica! Chemistry and Biochemistry, 
University of Innsbruck and Ludwig Boltzmann Institute of AIDS Research, Fritz Pregl StraBe 3, 
A-6020 Innsbruck, Austria 
Leif Dotevall, MD, Department of Infectious Diseases, Ostra Hospital, Goteborg University, 
S-41345 Goteborg, Sweden 
Thomas Gasse, MD, Department of Neurology, University of Innsbruck, AnichstraBe 35, 
A-6020 Innsbruck, Austria 
Lars Hagberg, MD, PhD, Department of Infectious Diseases, Goteborg University, same address 
Gernot P. Tilz, MD, professor, Department of Internal Medicine, University of Graz, 
Auenbruggerplatz 15, A-8036 Graz, Austria 
Gabriele Baier-Bitterlich, MD, Institute of Medica! Chemistry and Biochemistry, 
University of Innsbruck, same address 
Ulrike Demel, MD, Department of Interna! Medicine, University of Graz, same address 
Erich Schmutzhard, MD, professor, Department of Neurology, University of Innsbruck, same address 
Helmut Wachter, PhD, Institute of Medical Chemistry and Biochemistry, 
University of Innsbruck, same address 
acta dermatovenerologica A.P.A. Vol 5, 96, No 3-4 141