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Annals of the Rheumatic Diseases 1993; 52: 429-435
Urine neopterin as a parameter of disease activityin patients
with systemic lupus erythematosus:comparisons with serum sIL-2R and
antibodies todsDNA, erythrocyte sedimentation rate, andplasma C3,
C4, and C3 degradation products
K L Lim, A C Jones, N S Brown, R J Powell
AbstractObjectives-To investigate urine neop-terin as a
parameter of disease activity inan unselected group of patients
withsystemic lupus erythematosus (SLE) andto study the relation
between urineneopterin and certain patterns of organdisease and
differing drug regimens in thetreatment of SLE.Methods-Neopterin
was determined byhigh performance liquid chromatographyin 115 early
morning urine samples from68 patients with SLE. Serum
solubleinterleukin 2 receptor (sIL-2R) andantibodies to double
stranded DNA(dsDNA) were determined by enzymelinked immunosorbent
assay (ELISA),and the erythrocyte sedimentation rate(ESR), plasma
C3, C4, and C3degradation products (C3dg) weremeasured in
corresponding bloodsamples. Disease activity was scored usingthe
British Isles Lupus Assessment Group(BILAG) index.Results-Urine
neopterin was signifi-cantly increased in patients with activeand
inactive SLE compared with thecontrol group and was significantly
higherin patients with active than in those withinactive SLE. Urine
neopterin did notdistinguish between subsets of patientswith SLE
with particular patterns oforgandisease, as defined by the BILAG
index,nor was its level primarily influenced bydiffering drug
regimens. Levels of serumsIL-2R, antibodies to dsDNA, the ESR,and
plasma C3, C4, and C3dg were alsosignificantly different between
thepatients with active and inactive SLE.Unlike urine neopterin
there wasconsiderable overlap in the values oftheseparameters
between the two activitygroups. Highly significant
correlationsfound between urine neopterin and serumsIL-2R, ESR, and
plasma C3, C4, andC3dg suggest the close association ofneopterin
with clinical activity in SLE.Multivariate logistic regression
analysisshowed that urine neopterin >300 ,umol/mol creatinine
was a highly significantpredictor of disease activity with an
oddsratio of 3-51.
Conclusions-Determination of urineneopterin, a non-invasive,
relativelysimple and inexpensive measurement,appears to be the best
parameter forassessing and monitoring disease activityand treatment
in patients with SLE.
(Ann Rheum Dis 1993; 52: 429-435)
Numerous and varied abnormalities of theimmune system have been
reported in patientswith systemic lupus erythematosus (SLE).1
Inparticular, increases in circulating Tlymphocytes bearing the
activation markersHLA-DR and HLA-DP antigens,2 3the earlyactivation
antigen TLi-SA14 and membranebound interleukin 2 receptors (IL-2R)5
havebeen documented, supporting the concept ofTlymphocyte
upregulation. Particular attentionhas been focused on neopterin as
an indicatorof activation of the cellular immune system.6Neopterin
is a pyrazino-pyrimidine derivativeformed from guanosine
triphosphate within thebiosynthetic pathway of biopterin,7 but
unlikebiopterin, which is an important cofactor in anumber of
enzymatic reactions, thephysiological role of neopterin
remainsobscure. In vitro experiments have shown thatneopterin is
specifically produced by humanmacrophages when stimulated by
interferon -yreleased from activated T lymphocytes.8 9Evidence
exists that endothelial cells can be animportant source of
neopterin in vitro'0 but asyet endothelial cell production of
neopterin invivo has not been shown. Nevertheless,increased
neopterin has been shown to be anearly, specific and sensitive
marker ofactivation of the cellular immune system inseveral
clinical settings including allograftrejection, acute viral
intracellular bacterial andprotozoan infections, autoimmune
andinflammatory diseases, and certain malignantdiseases."lThe
erythrocyte sedimentation rate (ESR),
plasma/serum complement levels, andantibodies to double stranded
DNA (dsDNA)are accepted as measures of disease activity inSLE. Some
patients, however, may haveabnormalities in these tests for
considerableperiods yet show few clinical symptoms orfunctional
deterioration of a major organ,
Department ofImmunology,University Hospital,Queens
MedicalCentre, Nottingham,United KingdomK L LimR J PowellDepartment
of ClinicalChemistry, UniversityHospital, QueensMedical
Centre,Nottingham,United KingdomN S BrownDepartment ofRheumatology,
CityHospital, Nottingham,United KingdomA C JonesCorrespondence
to:Dr K L Iim,Clinical Immunology Unit,F Floor, University
Hospital,Queens Medical Centre,Nottingham NG7 2UTH,United
Kingdom.Accepted for publication1 February 1993
429
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Lim, J7ones, Brown, Powell
whereas others are markedly symptomatic withminor aberrations in
these test results. Somereports suggest that the measurement of
serumsoluble interleukin 2 receptors (sIL-2R),derived from surface
bound IL-2R in vivo, isa more reliable index of global clinical
activityin patients with SLE, specifically reflectingsubclinical
immune activity with respect toantibodies to dsDNA and complement
levels. 12This study investigates urine neopterin as aparameter of
disease activity in an unselectedgroup of patients with SLE, with
particularemphasis on the relation between urineneopterin and
certain patterns of organ diseaseand differing treatment
regimens.
Patients and methodsPATIENTS
Sixty eight patients attending the connectivetissue disease
clinics who fulfilled four or moreof the 1982 revised American
RheumatismAssociation (ARA) criteria for the classificationof SLE'3
were prospectively studied. A total of115 matched urine and serum
samples wereobtained from these patients over a period of12 months.
In addition, 65 apparently healthycontrols supplied urine samples
for analysisand 19 of the 65 healthy controls volunteeredserum
samples for estimations of sIL-2R (table1). None of the patients or
controls had aconcomitant viral or bacterial infection ordisorders
other than SLE at the time ofinvestigation. This study was approved
by theNottingham University Hospital ethicscommittee.The
computerised British Isles Lupus
Assessment Group (BIIAG) index'4 was usedto score SLE disease
activity by a singleobserver (KLL). The index consists of
86questions covering eight organ based systems,namely; general
mucocutaneous, nervous,musculoskeletal, cardiovascular,
vasculitis,renal, and haematological. A score of either 'A'or 'B'
in any of these organ systems wasconsidered to represent active
disease.
LABORATORY MEASUREMENTS
Determination of neopterin in early morningurine samples was by
reversed phase highperformance liquid chromatography asdescribed
previously'5 with minormodifications. In summary, urine
samples,protected from light and stored frozen, werecentrifuged to
remove debris, diluted one in 10with water containing
dimethylpterine as aninternal standard and injected directly onto
a
Table 1 Selected clinical and demographic features of the
control group and patients withsystemic lupus erythematosus
(SLE)
Features Control groups Patients with SLE(n=68)
Neopterin sIL-2R.(n=65) (n=19)*
Mean (SD) age (years)t 45 (12-6) 42 (10-6) 43 (12-8)Male/female
2/63 0/19 3/65Mean (SD) disease duration (years) 11*5 (9*5)Mean
number ofARA criteria fuilfilled* 5.4
*sIL-2R=soluble interleukin 2 receptor; ARA=American Rheumatism
Association.tp>0-05 by ANOVA.
Techsphere 5 ODS column (HPLCTechnology Ltd). A binary gradient
elutionwas used with an initial mobile phase of 2%methanol in 15
mmoVl phosphate buffer, pH6-4, increasing to 25% methanol after
12minutes, and neopterin was detected by itsnatural fluorescence
()ey 353 nm; Xem 438 nm).Creatinine was determined separately using
akinetic alkaline picrate (Jaffe) method andneopterin excretion was
expressed as pLmolImolcreatinine.Serum sIL-2R was measured using
a
commercially available sandwich enzymelinked immunosorbent assay
kit (Cellfree, TCell Diagnostics, Cambridge, MA, USA).Results were
expressed in U/ml relative to a setof standards supplied with the
kit.
Other laboratory determinants wereperformed by standard methods:
haemoglobinand white blood cell count with absolutedifferential
count using a Sysmex NE8000instrument; ESR by the Seditainer
ESRsystem; serum urea and electrolytes on anEktachem 700XR
instrument (KodakDiagnostic Ltd); plasma C3 and C4 bynephelometry;
plasma C3 degradationproducts (C3dg) by a double decker
immuno-diffusion method; and antibodies to dsDNAusing an ELISA kit
from DiamedicCorporation.
STATISTICAL PROCEDURE
Appropriate use of Student's t test andANOVA enabled comparisons
of age, diseaseduration, and number of ARA criteria
fulfilledbetween groups. For non-parametric studyvariables,
Spearman rank correlationcoefficients were computed for pairs
ofcontinuous data and the Mann Whitney U testwas used to compare
continuous variablesbetween two subgroups. When there weremore than
two subgroups the Kruskall WallisH test was used. To avoid
statistical bias, onlydata from the initial assessment of patients
whohad more than one assessment during thestudy period were
included in the correlationanalysis.Forward stepwise logistic
regression analysis
was chosen to identify the best predictor ofSLE disease
activity. The predictive variables(represented in binary format)
were ESR, C3,C4, C3dg, antibodies to dsDNA dichotomisedat 100 and
300 IU/ml, serum sIL-2Rdichotomised at 700, 750, 800, and 850
U/ml,and urine neopterin dichotomised at 200, 250,300, and 350
pLmol/mol creatinine. Abnormalresults were defined as ESR >10
mm/hour,C3
-
Urine neopterin as a parameter of disease activity in SLE
p < 0.0001
91370
S
I
05
*t
so
E
-J
E
a)
0
SD
de
I'
2400-
2000-
1600
1200-
800 -
400-
ConrogouInctve AcivControl group Inactive Active
(n = 65) (n = 60) (n = 55)
SLE patient samples
p < 0-05
I
0
S
I4
IS
* :0
: %-
* ~~~~I
_
Im
Control group Inactive Active(n = 19) (n = 60) (n = 55)
SLE patient samplesFigure 1 Urine neopterin and serum soluble
interleukin 2 receptor (sIL-2R) values in patients with active and
inactivesystemic lupus erythematosus (SLE) and their corresponding
control groups. The median values of each group is representedby
the solid line.
SERC and CYTEL) were used; p
-
Lim, _Jones, Brown, Powell
ip = 00006.
S.
0 T
1 0%
Inactive Acti0
Inactive Active
0-4 -
0-3 -
CD0)
0-2 -
0-1 -
0 -
p
-
Urine neopterin as a parameter of disease activity in SLE
* 13701300-tc
a 800--
o
o 600-E
zC 400-
200-.
0
t: *0 * *
~ ~ 0
* 0 .* .1
t *0 0041~~~~
400 800 1200 1600 2
Serum slL-2R (U/ml)Figure 3 Correlation between serum soluble
intreceptor (sIL-2R) and urine neoptenin in 68 patsystemtc lupus
erythematosus.
Table 2 Sensitivity, specificity and positive and negative
predictive values of leneopterin as a determinant of disease
activity in patients with systemic lupus ery
Urine neopterin levels Sensitivity Specificity Predictive
v(,umol/mol creatinine) (%) (%)
Positive
Active disease Inactive disease
>350 6350 42 77 62>300 6300 62 72 67>250 6250 71 57
60>200 6200 78 47 57
Kruskall-Wallis p = 0-024
Cytotoxicdrugs(n = 11)
-1370
Prednisolone Prednisolone Hydroxychloroquine/(n = 14) /Cytotoxic
untreated
drugs (n = 9)(n = 21)
Treatment regimensFigure 4 Medians (horizontal rule) and ranges
of urine neopterin concentratiosubgroups ofpatients with active
systemic lupus erythematosus receivingfour dif,treatment
regimens.
0 51 l0-0001I
in patients with activity in a single organ system(median 305,
range 91-1370) (p=0 0002), butno significant differences in urine
neopterinvalues were observed between subgroups ofpatients with
activity in a specific organ system(data not shown).
. DiscussionWe have shown a highly significant
associationbetween urine neopterin and disease activity ina group
of patients with diverse clinicalmanifestations of SLE.
Significantly increasedurine neopterin values were found in
patientswith active and inactive SLE compared with
0o0 24'00 healthy controls. Previously only patients with
renal lupus have been investigated,'7 in whomurine neopterin
correlated with SLE disease
terleukin 2 activity according to the Winfield criteria,'8
butitents with with the clinical severity of renal
disease. The raised urine neopterin values invels of urine
patients, with inactive SLE probably represent'thematosus
continuous low grade activation of the cellularPalues (Oo) immune
system without an association with
clinical symptoms or apparent deteriorationNegative in the
function of a major organ as defined
by the BIIAG index. Low grade cellularimmune activation in
inactive SLE is further
67 suggested by our serum sIL-2R data, which68 showed
significantly higher serum sIL-2R70
levels in patients with SLE than in controlsregardless of their
disease activity; this agreeswith other studies.'9-24 The
continuous cellularimmune activation may be
prognosticallysignificant.There was considerable overlap in
serum
sIL-2R values between the patients with activeand inactive SLE.
Serum sIL-2R values tendto be higher in active than in inactive
disease,however, and the difference in their medianvalues, though
small, remained significant atthe p
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Lim, _Jones, Brown, Powell
creatinine and plasma C4
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Urine neopterin as a parameter of disease activity in SLE
22 Huang Y, Perrin L, Miescher P, Zubler R. Correlation ofT and
B cell activities in vitro and serum IL-2 levels insystemic lupus
erythematosus. Immunol 1988; 141:827-33.
23 Semenzato G, Bambara L, Biasi D, et al. Increased serumlevels
of soluble interleukin-2 receptor in patients withsystemic lupus
erythematosus and rheumatoid arthritis.Clin Immunol 1988; 8:
447-52.
24 Manoussakis M, Papadopoulos G, Drosos A,Moutsopoulos H.
Soluble interleukin 2 receptormolecules in the serum of patients
with autoimmunediseases. Clin Immunol Immunopathol 1989; 50:
321-32.
25 Nelson D, Wagner D, Marcon L, et al. An analysis of
solubleIL-2 receptors in human neoplastic disorders. In:Albertini
A, Lentant C, Poeletti R, ed. Biotechnology inclinical medicine.
New York: Raven Press, 1987.
26 Manoussakis M, Germanidis G, Drosos A, MoutsopoulosH.
Impaired urinary excretion of soluble IL-2 receptorsin patients
with systemic lupus erythematosus andrheumatoid arthritis. Lupus
1992; 1: 105-9.
27 ter Borg E, Horst G, Limburg P, Kallenberg C. Changes
inplasma levels of interleukin-2 receptor in relation todisease
exacerbations and levels of anti-dsDNA andcomplement in systemic
lupus erythematosus. Clin ExpImmunol 1990; 82: 21-6.
28 Ward M, Dooley M, Christenson V, Pisetsky D. Therelationship
between soluble interleukin 2 receptor levelsand antidouble
stranded DNA antibody levels in patientswith systemic lupus
erythematosus. Rheumatol 1991; 18:235-40.
29 ter Borg E, Horst G, Hummel E, Limburg P, KallenbergC. Rises
in anti-double stranded DNA antibody levelsprior to exacerbations
of systemic lupus erythematosusare not merely due to polyclonal B
cell activation. ClinImmunol Immunopathol 1991; 59: 117-28.
30 ter Borg E, Horst G, Hummer E, Limburg P, KallenbergC.
Measurement of increases in anti-double-strandedDNA antibody levels
as a predictor of disease exacer-bations in systemic lupus
erythematosus: a long-term,prospective study. Arthritis Rheum 1990;
33: 634-43.
31 Niederwieser D, Fuchs D, Hausen A, et al. Neopterin as anew
biochemical marker in the clinical assessment ofulcerative colitis.
Immunobiology 1985; 170: 320-6.
32 Prior C, Bollbach R, Fuchs D, et al. Urinary neopterin,
amarker of clinical activity in patients with Crohn's
disease.ClinChimActa 1986; 155: 11-21.
33 Swaak A, Aarden L. Statius van Eps L, Feltkamp T. Anti-dsDNA
and complement profiles as prognostic guides insystemic lupus
erythematosus. Arthnitis Rheum 1979; 22:226-35.
34 Rokos H, Rokos K. A radioimmunoassay for determinationof
D-erythro-neopterin. In: Blair J A, ed. Chemistry andbiology of
pteridines. Berlin and New York: de Gruyter,1983: 815-9.
35 Werner E, Bichler A, Daxenbichler G, et al. Determinationof
neopterin in serurn and urine. Clin Chem 1987; 33:62-6.
36 Niederwieser A, Joller P, Seger R, et al. Neopterin in
AIDS,other immunodeficiencies, and bacterial and viralinfections.
Kin Wochenschr 1986; 64: 333-7.
435
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