Research Paper Mediators of Inflammation, 7, 309–312 (1998)

Corresponding Author Te l/Fax : (+30) 017 286 229 SERUM leve ls of IL-1 b , IL-6 and TNF-a were m easured in 48 healthy, te rm ed neonate s on the 1st (N1), 5th (N5) and 40th (N40) day after birth , com pared w ith th ose in m ate rnal se rum (MS), um bilical cord (UC) and adult controls . Cytokine value s in N1 and N5 were sign ifican tly e levated, th an those in UC and in con trols (P<0.0001). IL-1 b and IL-6 declined s ign ifican tly from N1 to N40 (P<0.0001), wh ile TNF-a in creased s ignificantly from N1 to N5 and declined thereafte r. MS ` IL-1 b and IL-6, but not MS ` TNF-a , w ere s ign ifican tly higher than those of con trols (P<0.0001). IL-1 b values depended on the m ode of de livery. In conclusion , th e in creased concen trations of IL-1 b , IL-6 and TNF-a durin g th e pe rin atal pe riod m igh t suggest th eir involvem ent in an in flam m ation like proces s durin g norm al parturition, and re fle ct also a newborn im m une re sponse to th e s tr es s of de livery and environm ental changes .


Introduction
The inflammatory c ytokine s, inte rleukin-1b (IL-1b ), inte rleukin-6 (IL-6) and tumour ne crosis fac tor-a (TNF-a ) are imp ortant me diators of host re sponse to stre ss and infection. All three cytokines have alre ady be e n found in c ells, tissues and fluids as sociated w ith pre gnanc y. 1 -5 It has be en hypothesized that IL-1b may play a signific ant role in the e mbryoge ne sis, de ve lopme nt and diffe re ntiation of the fe tus by re gulating the grow th and differe ntiation of tissues and/or organs. 2 More ove r, IL-1b , stimulating prostaglandin biosynthesis by intraute rine tissues, may signal the onset of labour and also strongly upre gulate IL-6 production. In te rm pre gnancy IL-6 may orchestrate biochemic al, immunologic al and physiological change s that contribute to maternal and fetal survival. 3 In addition, TNF-a , dete c te d in the placenta, amniotic fluid and decidua, may contribute to the re gulation of ce ll prolife ration at the uteroplace ntal unit. 6,7 Ele vated value s of IL-1b , IL-6 and TNF-a during both te rm and pre te rm labour have already be e n re porte d in amniotic fluid samples, 2,3 ,8 -1 1 in mate rnal se rum 7 ,1 2 -14 and in c ervicovaginal se cre tions; 15 -1 6 but, little is know n about the pre se nce of the se inflammatory cytokines in e arly ne onatal life . For this re ason, our aim w as to study the c onc entrations of IL-1b , IL-6 and TNF-a in ne w born infant serum on the first days after birth, c ompare d w ith those in mate rnal and umbilic al cord serum during parturition , in order to inve stigate the changes of all thre e inflammatory cytokines during the pe rinatal period.

Patients and methods
This study w as approved by the Ethic s Committe e of the Are te ion Te aching Hosp ital. Follow ing informe d maternal c onsent, peripheral blood samples w ere collecte d from 48 healthy, te rmed ne onates (clinical charac te ristics in the Table 1), during the 1s t (N1), the 5th (N5) and the 40th (N40) days afte r birth. In 25 of these cases, mate rnal se rum (MS) during the first stage of labour and umbilical c ord (UC) se rum in the se cond stage of labour w ere also obtained. Serum samples from 25 healthy adults w ere also analyse d simultaneously, as c ontrols.
Clinical ex amination of ne onates, their mothers and controls, as w ell as serum re active prote in (CRP) dete rmination s w ere performed and all subjec ts w e re judged infection-free .
Blood samples, c ollec te d in pyrogen-free tubes, w ere c entrifuged at onc e afte r clotting and store d aliquotte d in -30°C until as say. All thre e cytokine s Data w ere analysed using non parametric methods, as the y did not show a normal distribution (Kolmogorov-Smyrnov te st). A Wilc ox on paired or non paired te st, as appropriate, w as used in comparing the distribution of c ytokines in the diffe re nt serum samples, w hile Kruskal-Wallis ANOVA w as used to compare the cytokine values among the three groups ac cording to the mode of delivery. Correlations w ere evaluated w ith Spearman rank c orre lation coefficie nts.

Results
Cytokine conc entrations, as median value s and range s, in the diffe re nt serum samp le s are given in Table 2.  Umbilical c ord values of TNF-a w e re significantly higher than those in controls (P< 0.0001), but low er than in N40. The UC value s of IL-1b and IL-6 w ere highe r, not significantly though, than those in adult controls and in ne onatal sample s N40. d Maternal se rum IL-1b and IL-6, but not TNF-a value s w ere significantly highe r than those in c ontrols (P< 0.0001). d Maternal se rum IL-1b values w e re also significantly higher than those in N1 (P<0.0001), w hile those of IL-6 and TNF-a w ere significantly low er (P< 0.0001).

Discussion
The patte rns of inflammatory cytokines IL-1b , IL-6 and TNF-a in ne w born infants during the p erinatal period demonstrate d signific ant change s in cytokine values, re lated to the time after birth. TNF-a pre se nte d significantly elevate d umbilic al c ord value s, c ompare d w ith those in controls and in maternal serum, w ith a significant incre ase in ne onatal se rum on the 1st and further on the 5th day after birth, declining slow ly there after. The c oncentration s of IL-1b and IL-6 in umbilical cord w e re also highe r, marginally though, than those in controls, inc re as ing markedly further on the 1s t day after birth and de clining there after, until normalization 40 days later.
The se data, c oncerning e levated values of all thre e inflammatory c ytokines in healthy ne w born infants, are the ne w findings of this study and may p ossibly re flec t an immune re sponse of ne onate s to the stre ss of de livery and environme ntal influenc es during the early ne onatal life . This hypothesis is also supp orte d by the de pendenc e of IL-1b value s in UC and N1 on the mode of delivery and the elevation of cytokine values in UC, mainly of TNF-a , compared w ith those in controls.
To the be st of our know le dge , serum le ve ls of the se inflammatory c ytokine s in healthy, non-infecte d ne onates during the early ne onatal pe riod are re p orte d he re for the first time. Sinc e all these pre gnanc ie s w ere une ve ntful and le d to the birth of healthy, te rmed ne w borns, the se le vels may be considere d to be a good re flection of the normal range. Mediators of Inflammation · Vol 7 · 1998 It has be en hypothesized that the pre se nce of inc re as ed TNF-a value s in p yar and bre as t milk may induc e the developmental and maturational p roc esses of the immune syste m in ne onates. 1 7,18 How ever, our TNF-a values in umbilic al cord show e d a significant ele vation already during birth, be fore bre ast fee ding, stre ngthening our suggestion, that normal te rm labour itself, might constitute an inflammation-like proce ss, that promote s the ac tivation of the ne w born immune syste m.
Similarly, w e have already re ported e levated serum values of soluble inte rleukin-2 re ce ptors (sIL-2R) in umbilical c ord and in serum samples from healthy te rm ne onate s on the 1st and 5th < day after birth, inc re as ing progre ssive ly from the 1st to the 3rd sample and strongly depe nde nt on the mode of delive ry. 1 9 The ele vation of this c ytokine , a characte ristic marke r of immune activation, w as attributed to the dynamic ex pansion of the ne onatal immune syste m, in re spons e to e nvironmental changes, during and after birth in the e arly ne onatal period. This phenomenon w as also re flec te d in the elevation of othe r grow th factors, as w e have p re viously re p orte d for angiogenin le ve ls in the pe rinatal period. 2 0 We have re c ently also found, how eve r, soluble inte rcellular adhe sion mole cule-1 (sICAM-1) levels in ne onatal serum sample s on the 1s t day of life to be low er than those in controls, though incre asing progressively there after and ex ce eding those of controls 1 month afte r birth. 21 The se diffe re nc es in ex pre ssion of cytokines during parturition may be due to their diffe re ntiate d role s in the cytokine casc ade during inflammation and immune proc ess.
More over, in agre ement w ith pre vious re ports, 7 ,1 8,2 2 -24 this study de monstrated ve ry inc re as ed values of IL-1b and IL-6, but not of TNF-a , in maternal c irc ulation during the first stage of labour. The value s of IL-1b w e re strongly re late d to the mode of delivery, a finding cons is te nt w ith the dominant role of this cytokine in the initiation of parturition. [1][2][3] The e levation of mate rnal serum IL-1b and IL-6 values se ems to re fle ct a syste mic re action in the mother to he r fe tus and might be analogous to the cytokine inc re as e observe d in the se rum of re nal transplant re c ipie nts, be fore ac ute re je ction of the graft. 25 The final inc re ase at the onse t of labour c ould be a similar 'sign of re jec tion'. 7 Another p ossible ex planation might be the physical e ffort of giving birth, be cause prolonge d ex ercis e is as sociated w ith incre ased produc tion of IL-6 from stimulated blood mononucle ar ce lls. 26 In contrast, the low value s of TNF-a in mate rnal se rum sugge st a paracrine action for this cytokine in maternal and fe tal tissue s, as the y re gulate the grow th of trophoblast during pre gnancy and contribute to the immune proce sse s, as sociated w ith labour. 2 4 Ge ne rally, IL-1b , IL-6 and TNF-a re lease d by chorion, amnion and de cidua and ac ting on these mem-branes, mate rnal se rum c ytokine value s re flec t functions that take place in the gestational tissues and depend on the pe rmeability of these tissue s by the se prote ins. Thus, our ele vated mate rnal serum IL-6 and low TNF-a are in agre ement w ith a pre vious re port, 2 7 re garding the perme ability of fe tal membranes by these c ytokines. In contrast, our re sults on incre ase d maternal se rum IL-1b values do not agree w ith the latter study.
In c onc lusion, the pre senc e of incre ase d inflammatory cytokines IL-1b , IL-6 and TNF-a during the e arly ne onatal period (a) suggests their contribution to the immune syste m alte rations that may signal or propagate the onse t of parturition and (b) re fle cts also a ne onatal immune re sponse to the stress of de livery and environmental changes in the first days afte r birth .