it is well established that high - risk ( hr ) human papillomavirus ( hpv ) types are causative 
 for the development of cervical cancer [ 13 ] .
the majority of hpv infections 
 are cleared without further consequences for the host , but some infections with 
 hr - hpv types may give rise to high - grade cervical intraepithelial neoplasia 
 ( cin iii ) and cervical cancer [ 46 ] .
there is evidence that cell - mediated 
 immune responses of the host , both systemic and local , are important 
 determinants for the course of the infection .
cell - mediated immune responses 
 are regulated by t lymphocytes [ t - helper ( th ) lymphocytes and cytotoxic 
 lymphocytes ( ctls ) ] in cooperation with antigen - presenting cells ( apcs ) 
 [ monocytes ( mcs ) and dendritic cells ( dcs ) ] .
these cells all release cytokines 
 that can influence one another 's synthesis and actions in the setting of an 
 immuno - regulating cytokine network .
cytokines in immune responses to infection 
 are often classified as immuno - stimulating ( tumour - suppressing ) th1type cytokines 
 and immuno - inhibitory ( tumour - promoting ) th2-type cytokines .
th1-type cytokines 
 such as interferon  ( ifn ) , 
 tumour necrosis factor  ( tnf ) , 
 interleukin 2 ( il-2 ) , and il-12 are produced mainly by lymphocytes , apcs , and 
 natural killer cells ( nk - cells ) .
th2-type cytokines ( il-4 , il-5 , il-6 , il-8 , il-10 ) , produced by 
 lymphocytes and mcs , are immuno - inhibitory for cell - mediated responses and 
 predominantly induce humoral immunity [ 8 , 9 ]
. qualitative and quantitative 
 analyses of cytokine profiles have been used to characterize the immune 
 response in hpv - related cin .
these were performed with peripheral blood 
 mononuclear cells ( pbmcs ) [ 1012 ] or with t - cell fractions isolated from 
 pbmcs [ 1316 ] and occasionally with whole blood cultures   after 
 stimulation with several antigens .
selective cytokines , mostly ifn [ 11 , 12 , 1418 ] , il-2 
 [ 1014 ] ; and occasionally the apc - derived il-12   or tnf   were measured together with one or two of 
 the typical th2-type cytokines il-4 , il-5 , and il-10 [ 11 , 12 , 16 , 17 ] . generally 
 a shift from a th1-type to a th2-type cytokine response was observed when healthy 
 controls or women with low - grade squamous intraepithelial lesions ( lsil ) were 
 compared with cases of high - grade sil ( hsil ) or cervical 
 carcinoma [ 7 , 11 , 17 , 19 ] . we 
 previously observed manifestation of a th2-type cytokine pattern in plasma of 
 hr - hpv - positive women during carcinogenesis of cervical cancer at the stage of 
 cin iii .
recent studies with isolated t - cell fractions stimulated with 
 hpv16-derived oncopeptides indicate a reactivation of an inflammatory response 
 in patients with carcinoma [ 12 , 15 ] .
these results let us assume that 
 significant changes in the immunocompetence of circulating leukocytes are 
 involved in the development from cervical dysplasia to cervical cancer . in the 
 present study we used whole blood cultures from hr - hpv - negative controls , 
 hr - hpv - positive women without cervical dysplasia and hr - hpv positive patients 
 with different grades of cin and cervical cancer to investigate changes in 
 immunocompetence expressed in the capacity of circulating leukocytes to release 
 cytokines in response to a mitogenic challenge . of interest
were the effect of 
 hr - hpv infection without clinical manifestations , the special position of cin 
 iii with a th2-type cytokine response , and a possible revival of inflammatory 
 cytokine activity in cervical carcinoma .
inclusion took 
 place at the outpatient clinic of the obstetrics and gynaecology department of the erasmus 
 university medical center ( rotterdam , the netherlands ) between july 2000 and august 2002 .
our selection of patients for this study was 
 based on the presence of hr - hpv and the grade of cervical intraepithelial 
 neoplasia .
histology results were defined as no dysplasia , mild 
 dysplasia ( cin i ) , moderate dysplasia ( cin ii ) , severe dysplasia ( cin iii ) , or 
 ( micro- ) invasive cancer . an experienced pathologist revised all histological 
 samples .
women with cin i lesions ( mild dysplasia ) were excluded since more 
 than fifty percent of our patients with cin i turned out to be hr - hpv - negative . 

healthy women who attended the outpatient clinic for a regular sterilisation 
 procedure were recruited as hr - hpv - negative controls after sampling for 
 histology and hpv .
exclusion criteria for all participants were ( anamnestic 
 required ) : postmenopausal state , pregnancy at time of sampling , chronic 
 diseases ( diabetes , allergy , auto - immune ) , presence of sexually transmitted 
 diseases ( stds ) and infection with human immunodeficiency virus ( hiv ) , signs of 
 acute infection at time of sampling , and an immune - compromised state . with the 
 exception of oral contraceptives , no participant used medication on a regular 
 base . no participant had used pain - medication ( including nsaids ) for at least 
 two weeks prior to sampling in order to avoid the well - known influence of 
 nsaids on cytokine release from pbmcs .
the study protocol was approved by the 
 ethics committee of the erasmus medical center and all women voluntarily gave 
 signed informed consent .
cervical scrapes 
 for hpv detection and typing were taken using a cervical bio - sampler ( accellon 
 combi medscand medical , malm , sweden ) .
hpv testing was performed with the consensus 
 gp5+/gp6 + pcr enzyme immunoassay ( eia ) using a cocktail probe covering 37 
 ( sub- ) types , including all ( probably ) hr - hpv types , as previously 
 described .
we used -globin pcr to identify sampling errors and to monitor 
 for pcr inhibitors .
additionally , reverse line blot ( rbl ) analysis was 
 performed on pcr - eia - positive cases to identify individual hpv types . for the preparation of whole blood cultures , 

peripheral venous blood samples were collected between 812 am 
 in sterile endotoxin - free vacutainers ( endo tubes chromogenix ab , mlndal , sweden ) 
 coated with na - heparin as anticoagulant , and immediately processed . for a leukocyte count peripheral venous blood 
 samples , collected between 8 and 12 am , were drawn into endotoxin - free 
 vacutainers ( becton - dickinson , meylan , nj , usa ) with ethylene - diaminetetra - acetic acid ( edta ) 
 as anticoagulant and leukocyte counts performed with a sysmex xe-2100 . for preparation of whole blood cultures ,
blood 
 was diluted 1:10 with rpmi 1640 culture medium with 25 mm hepes , supplemented 
 with 10 u / ml penicillin , 100 g / ml 
 streptomycin , and 4 mm l - glutamine ( medium and supplements from life 
 technologies bv , breda , the netherlands ) .
diluted blood was distributed in cell 
 culture plates and incubated with phytohemagglutinin ( pha ) ( sigma - aldrish , 
 mo , usa ) dissolved in rpmi medium to a final concentration of 10 g / ml blood culture , for 96 hours at 37c 
 and 5% co2 .
all cultures were sampled at 0 , 
 24 , 48 , 72 , and 96 hours , centrifuged for 10 minutes at 4c and 1500 g , and culture supernatants kept at 80c 
 until analysis .
all samples were 
 analysed by commercially available enzyme - linked immunoassays ( biosource 
 europe , nivelle , belgium ) for the cytokines tnf , ifn , 
 il-2 , il-4 , il-10 , and il-12 .
the detecting antibody in the immunoassay for 
 il-12 recognized the bioactive heterodimeric ( p40 + p35 ) cytokine as well as the 
 subunit p40 monomer or homodimer . according to the manufacturer , the minimal 
 detectable concentrations ( mdcs ) and intra- and interassay coefficients 
 ( cvs ) of variation were as follows : tnf : mdc , 3 pg / ml ; cvs , < 6 and < 10% ; ifn : mdc , 2 pg / ml ; cvs , < 5 and < 10% ; il-2 : 
 mdc , 7 pg / ml ; vcs , < 6 and < 10% ; il-4 : mdc , 2 pg / ml ; cvs , < 5 and < 7% ; il-10 : mdc , 1 pg / ml ; cvs ,
< 5 and < 10% ; il-12 + p40 : mdc , 1.5 pg / ml , 
 cvs , < 10 and < 10% . preliminary 
 komolgoroff - smirnov tests showed an abnor mal distribution of cytokine values 
 in pha - stimulated whole blood cultures .
accordingly , cytokine data are 
 presented as medians with ranges unless stated otherwise . the nonparametric 
 kruskal - wallis test ( k. w. test ) and mann - whitney 's u - test were used as 
 appropriate to assess differences in cytokine levels between groups .
levels of statistical significance were adjusted 
 for the number of comparisons according to bonferroni 's method , as indicated in 
 the graphics .
differences in patient characteristics between groups were 
 evaluated by one - way anova and unpaired two - tailed t - tests .
spearman 's correlations were used to 
 investigate possible relations between age at time of sampling and released 
 cytokines .
five of them were excluded because of diabetes ( n=1 ) , 
 allergy ( n=2 ) , autoimmune disease ( n=1 ) , or acute infection at time of sampling 
 ( n=1 ) , leaving 30 women eligible for inclusion : 10 women with moderate 
 dysplasia ( cin ii ) , 10 women with severe dysplasia ( cin iii ) , and 10 women with 
 cervical carcinoma ( 8 squamous cell carcinoma , 2 adenocarcinoma ) .
all women of 
 this group revealed a positive gp5+/6 + hr - hpv pcr test .
three of them were excluded because of the 
 presence of allergy ( n=2 ) or acute infection at time of sampling ( n=1 ) , leaving 
 19 healthy women without cervical dysplasia .
nine women had a positive hr - hpv 
 test , 10 women tested negative for hpv - dna , forming the control group .
baseline 
 characteristics of the study groups are summarized in table 1 . the mean age of hr - hpv - positive women without cervical dysplasia is 
 significantly lower than in the other groups .
this could be expected since 
 first infection without clinical manifestation is frequently observed in young 
 sexually active women .
spearman 's correlations between age at time of 
 sampling and released cytokines over the whole group of patients and controls 
 were not significant ( data not shown ) .
the changes in immune - competence in our 
 study are not related to age .
the results of cytokine 
 assays were calculated per 10 leukocytes , in order to stratify for 
 possible different numbers of cytokine - producing leukocytes between study 
 subjects [ 20 , 24 ] .
preliminary 
 experiments were carried out on all investigated cytokines to determine the 
 time of peak production in response to pha stimulation of 
 our whole blood culture system ( data not shown ) .
cytokine concentrations from 0 
 to 96 hours stimulation time were analysed in at least six randomly chosen 
 study subjects for each stage of cin .
peak time for tnf , ifn , and 
 il-12 + p40 production was 72 hours , for il-2 48 hours of cultivation time . 

a typical 
 sample for the time - course of cytokine release in our blood culture system is 
 shown in figure 1 . in 
 general
our data of maximum cytokine release are in accordance with kinetic 
 studies of pbmc 's . on the basis of these results ,
il-2 release was 
 determined after 48 hours , release of tnf , ifn , and 
 il-12 + p40 after 72 hours ; and of il-4 and il-10 after 48 and 72 hours of 
 cultivation . for calculations of the latter two cytokines values of maximal 
 release
was observed between the two groups of women without 
 dysplasia : with the exception of il-12 all investigated cytokines were 
 significantly increased in hr - hpv - positive women .
the results are summarized in 
 table 2 . in hr - hpv - infected women , 

release of th1-type cytokines ifn , tnf , and il-2 decreased with increasing 
 grades of cin .
il-12 reached a 
 maximum in cin ii and decreased in cin iii and carcinoma ; but the differences 
 between groups were statistically not significant ( k. w. test : p=.068 for il-12 + p40 , 
 p=.264 for ifn , p=.077 for tnf and p=.071 for il-2 ) .
release 
 reached a maximum for il-10 and il-4 in patients with cin iii and decreased 
 significantly for both cytokines in patients with invasive carcinoma . 

the results are summarized 
 in figure 2 . in order to characterize a possible th1-type / th2-type shift we calculated the ratios of th1-type cytokines il-12 , ifn , tnf , and 
 il-2 to th2-type cytokines il-10 and il-4 in hr - hpv infected groups .
( results of 
 k. w. tests : il-12/il-10 p=.005 , il-12/il-4 p=.01 , ifn/il-10 p=.013 , ifn/il-4 p=.015 , tnf/il-10 p=.303 , tnf/il-4 p=.096 , il-2/il-10 p=.642 , il-2/il-4 p=.251 ) .
there was a significant decrease in th1-type / th2-type ratios between cin ii and cin iii for il-12/il-4 and il-12/il-10 .
also , ifn/il-4 and ifn/il-10 showed a similar though statistically 
 not significant trend as demonstrated in figure 3 .
this increase was significant for il-12/il-4 , 
 il-12/il-10 , ifn/il-4 , and ifn/il-10 . in order to characterize a possible th-1 type 
 cytokine pattern after establishment of an invasive carcinoma we compared 
  cytokine levels in pha - stimulated blood cultures of patients with invasive 
 carcinoma with levels in hr - hpv - positive women without dysplasia .
there was no 
 difference between levels of il-12 + p40 and ifn in both groups , but release of tnf and 
 il-2 as well as of il-10 and il-4 was significantly lower in patients with 
 carcinoma .
the significant 
 increase in th1-type as well as th2type cytokines in 
 our hr - hpv - positive women with normal histology suggests viral activation of 
 the systemic cytokine network and induction of cell - mediated immunity after 
 initial hr - hpv infection ( table 2 ) . to our knowledge
this is the first 
 description of activation of the systemic cytokine network in hr - hpv - positive 
 women without dysplasia .
cytokine release 
 changed to an antiinflammatory , tumour - promoting pattern by increase in il-4 
 and il-10 expression at the stage of cin iii .
this result confirms and extends 
 our earlier observations of a change to a th2-type cytokine pattern in the 
 circulation of patients with cin iii   and is in agreement with earlier 
 studies showing a shift from th1-type to th2-type cytokines during 
 carcinogenesis .
. 
 observed decreased ifn and 
 il-2 and increased il-4 and il-10 in mitogen - stimulated cultures of pbmcs 
 isolated from women with cin iii when compared with cultures from hr - hpv - negative women .
described increased il-10 and decreased il-12 
 release in whole blood cultures of patients with hsil when compared with hr - hpv - negative controls .
they found decreasing il-2 
 release with increasing severity of the disease , which is in agreement with our 
 results for il-2 .
the observed minimium for ifn release in cin iii but not in invasive 
 carcinoma differs from the observations of an earlier study by mori et al . 
  where pha - stimulated ifn 
 release from pbmcs in cases of invasive carcinoma was significantly decreased 
 when compared with data from healthy women . in the study of mori et al . 

however , the presence of hr - hpv was not investigated , which might explain the 
 difference in results with our study .
a shift to a 
 th2-type cytokine pattern in cin iii was more obvious when the ratios between 
 th1-type and th2-type cytokines ( figure 2 ) are evaluated .
they show a 
 tumour - promoting change in cytokine balance , significant for il-12/il-4 and 
 il-12/il-10 , and a trend for ifn/il-4 , ifn/il-10 , and tnf/il-4 .
our study describes for the first time changes in the cytokine pattern 
 within the cytokine network , developing from hr - hpv infection without clinical 
 symptoms via cin ii and cin iii to carcinoma .
il-12 is one of the 
 first cytokines released during an innate immune reaction and stimulates a th-1 
 type cytokine response in cell - mediated immunity .
our hr - hpv - positive women 
 with normal histology demonstrated significantly increased th1- and th2-type cytokine release , with the 
 exception of il-12 which was low .
our observation of high secretion of il-12 in 
 cin ii might be explained by an observation made by moscicki et al . .
these 
 authors reported high levels of il-12 in cervical mucous in hsil and 
 hypothesized that high il-12 levels could represent a defence mechanism in 
 turning on a th1-type antitumour response and , as il-12 is known to inhibit 
 angiogenesis , preventing growth of a tumour .
the significant 
 increase of the four cytokine ratios between cin iii and carcinoma may indicate 
 that the presence of a tumour with an inflammatory reaction and exposure of 
 viral antigens ( high viral load ) eventually induces a certain t - cell response . 

this response remains incomplete as shown in our cytokine data presented in 
 table 3 .
values of ifn and il-12 release in cervical carcinoma are comparable 
 to data obtained after initial hr - hpv infection ; all other cytokine levels 
 remain significantly lower .
these results suggest a second deregulated and incompetent 
 immune response in cervical carcinoma , probably due to manifestation of an 
 inflammatory effect of the tumour itself .
this reaction is partly comparable to 
 the inflammatory reaction on the initial hr - hpv infection , as expressed in the 
 ratio 's of ifn and il-12 in figures 3(a ) , 3(b ) , 3(e ) , 3(f ) .
these results are in 
 agreement with observations of de jong et al . , and steele 
 et al .  
studied t - cell responses to hpv 16 oncoproteins by measuring ifn release in women with low- and high - grade cin and 
 cervical carcinoma and found higher levels of t - cell responses in carcinoma 
 patients compared to high - grade cin cases .
a similar observation was made by de 
 jong et al . who investigated hpv16-positive women .
this study reports a 
 higher frequency of hpv16-specific cd4 + t - cell responses in patients with 
 cervical carcinoma than in women with cin iii lesions .
the increase in the ifn/il-4 ratio found in our study was not observed by de jong et al .  
when 
 t - cell cultures were stimulated with pha . in part , this discrepancy might be 
 owing to differences in hr - hpv types within the study groups since de jong et 
 al .
correlations between specific 
 hpv types and ifn 
 release , possibly influenced by ifn gene polymorphisms , are suspected but not yet 
 fully investigated . it was our goal 
 to study changes in the cytokine network in blood of hr - hpv - infected women at 
 various stages of cin upto onset of cervical carcinoma .
the use of whole blood 
 cultures for determination of mitogen - stimulated cytokine release by 
 immunocompetent leukocytes has distinct advantages over cultures of isolated 
 leukocytes or lymphocytes .
it permits interaction between different leukocytes , 
 preserves concentrations of stimulatory and inhibitory mediators , and avoids 
 activation and changes in cell ratios associated with procedures of isolation 
 and purification . for stimulation of 
 the cytokine network we chose the mitogen pha .  
pha activates mainly lymphocytes 
 and induces rapid cell proliferation together with release of inflammatory and 
 immune cytokines .
endotoxin ( lps ) as used in jacobs ' study   induces mainly 
 inflammatory cytokines but almost no lymphocyte - derived interleukins .
most studies dealing with cytokine patterns in hr - hpv - related 
 cervical neoplasia and cancer concentrate on infections with hpv 16 ( the most 
 frequently observed oncogenic hpv type in caucasian population ) .
in contrast 
 to these studies we did not select our patients for particular hr - hpv 
 types .
the small sample size of our study groups did not allow us to correlate 
 cytokine response with specific hr - hpv - types .
further studies with enlarged 
 numbers of participants are needed to investigate the individual impact of 
 different hr - hpv - types on the cytokine   network .
1 . our study suggests that infection with hr - hpv in women without 
 cervical dysplasia induces activation of the cytokine network .
2 . manifestation of a tumour induces a second deregulated and 
 incompetent immune response .
3 . our 
 results confirm and expand our earlier observations on circulating cytokines : 
 significant changes in the kinetics of cytokine release to a th2-type immune 
 response in blood of women with cervical dysplasia occur progressively from cin 
 ii to cin iii .
these 
 immunological findings are supported by clinical observations : many cin i or ii 
 lesions usually regress without treatment , whereas cin iii lesions mostly will 
 develop into invasive cancer if not properly treated .
 
 aims . we investigated the effect of hr - hpv infection on the capacity of the cytokine network in whole blood cultures during carcinogenesis of cervical carcinoma . 
 
 methods . 
 thirty - nine women with moderate dysplasia , severe dysplasia , cervical carcinoma , or without dysplasia formed the study group . 
 the control group consisted of 10 hr - hpv - negative women without cin . 
 whole blood cultures were stimulated with phytohemagglutinin ( pha ) and concentrations of tumour necrosis factor  ( tnf ) , interferon  ( ifn ) , 
 interleukin 2 ( il-2 ) , interleukin 12 ( il-12 ) , interleukin 4 ( il-4 ) , and interleukin 10 ( il-10 ) were determined by elisas . 
 results . 
 a significant increase in cytokine release was detected in hr - hpv - positive women without dysplasia . in women with cervical cancer , 
 release of ifn and il-12 was of the same magnitude as in hr - hpv - positive women without clinical manifestations . 
 most th1-type / th2-type ratios decreased form cin ii to cin iii , and increased from cin iii to invasive carcinoma . conclusions . 
 ( 1 ) infection with hr - hpv without expression of cervical dysplasia induces activation of the cytokine network . 
 ( 2 ) increases in ratios of th1-type to th2-type cytokines at the stage of cervical carcinoma were found by comparison with stage cin iii . 
 ( 3 ) significant changes in the kinetics of cytokine release to a th2-type immune response in blood of women with 
 cervical dysplasia occurred progressively from cin ii to cin iii . 
1. INTRODUCTION
2. MATERIALS
AND METHODS
3. RESULTS
4. DISCUSSION
5. CONCLUSIONS