003 I-3998/93/3304-0380a03.00/0 PEDIATRIC RESEARCH Vol. 23. No. 4. 1993 Copyright O 1993 International Pediatric Research Foundation. Inc. Prrnrc~drn C'. S..4.

Tumor Necrosis Factor-a, -16, and Interleukind Plasma Levels in Neonatal Sepsis

E. S. J. M. DE B0NT.A. MARTENS. J. VAN RAAN. G. SAMSON. W. P. F. FETTER. A. OKKEN. AND L. H. F. M. DE LEIJ Department of pediatric.^, Divi.sion of Neonatolog~,[E.S.J.hf.d. B.. U: P. F. F.], and Department c?f Clinical Chemi.stry /..1. I\{.. J. I '. R.. G. S.1. Sophia Ho.spita1. Zu,olle: and Department c?f Pediatrics. Division r?/' Neonatologj. /.4.0./, and Department c?fClinical Itnm~cnolo~~~[LH.F.h4.d. L.]. C:niversitj,Ho.spita1 Groningen. Groningen. The Netherlands

ARSTRAn. -a, IL-18, and IL-6 induce severe hypotension and fever in animals and humans are thought to be involved in the pathogenesis of sepsis ( 16- 19). Similar effects have been observed when TNFa is given with gram-negative bacteria. We studied these (6. 20-23). It is suggested in the literature that IL-1P potentiates during neonatal sepsis with mainly gram-positive bacteria. the effects of TNFa several-fold (24, 25). Ten newborns with clinical sepsis and 22 healthy controls Neonatal sepsis is a disease with a high morbidity and mortality were enrolled in the study. TNFa plasma levels proved to and is mainly caused by gram-positive bacteria (26). Because be increased in the newborns with sepsis up to 560 f 234 these microbial species are infrequently associated with infections pg/mL (ng/L) versus 36 f 4 pg/mL (ng/L) in the control in adults. their occurrence may be explained by the immature group (p < 0.005), whereas IL-6 plasma levels in newborns immune system in newborns (27). Cell wall fragments and toxins with sepsis were 79.700 f 37.500 pg/mL (ng/L) versus 55 from gram-positive organisms can induce TNFa and IL- I P pro- f 28 pg/mL (ng/L) in the control group (p < 0.01). The duction (28-30). In agreement with this, a recent study indicates IL-IB plasma levels were only slightly elevated in the group that TNFa serum levels are high in newborns with systemic newborns with sepsis [up to 18 f 5 pg/mL (ng/L) versus 7 infections and shock (3 I). 5 1 pg/mL (ng/L) in the control group (p < 0.01)). After The aim of the present study is to seek a correlation between the start of therapy with antibiotics, both TNFa and IL-6 the plasma levels of TNFa, IL-IP, and IL-6 and the clinical plasma levels decreased concomitantly with the improve- phenomena observed during neonatal sepsis. ment of the clinical situation within 2 d. These data confirm the abundant presence of TNFa and IL-6 during neonatal MATERIALS AND METHODS sepsis, whereas IL-1B appeared to be present in small amounts only. Nevertheless, the IL-18 but not the TNFa EDTA blood (EDTA, 1.5 mg/mL blood) specimens were plasma level appeared to correlate inversely with the de- obtained by venipuncture or from an arterial catheter and im- crease in diastolic tension as standardized according to mediately transported on ice to the laboratory. Plasma was birth weight (R = 0.66, p = 0.04). TNFa, IL-18, and IL- separated from the blood within 30 min. Aliquots were stored at 6 were not correlated with any febrile response in the group -80°C until assayed. The plasma TNFa, IL-ID, and IL-6 levels with sepsis. Inasmuch as only moderate temperature in- were measured using enzyme-linked immuoassays from Medge- creases were seen in these patients, we hypothesize that a nix according to the manufacturer's procedure (EASIA-assay, low IL-I@ plasma level may explain the lack of a febrile Medgenix, Brussels. Belgium). Because of the smaller volume of response during neonatal sepsis. (Pediatr Res 33: 380- the blood specimens that can be taken from neonates, we used 383,1993) an adapted method with smaller sample volumes (100 pL for TNFa and IL- I P measurements and 50 pL for the IL-6 meas- Abbreviations urement). The control, interassay, and intraassay values obtained with this procedure were within the same range as with the TNFa, tumor necrosis factor a original method. The detection limit for TNFa was 25 pg/mL (ng/L); for IL-IP, 4 pg/mL (ng/L); and for IL-6, 6 pg/mL (ng/ L). In the septic group. the first blood sample was taken before the start of antibiotic treatment, and the following samples were taken every 8 to 12 h over 2 to 3 d. In the control group, one TNFa, IL- I P, and IL-6 are considered to be important media- sample was taken. tors of the systemic host response to infection (1-6). These Informed consent was obtained from the parents of each cytokines are produced mainly by activated and newborn before the start of the study. The study was approved macrophages (7-9). During gram-negative sepsis in both children by the hospital ethics committee. and adults, TNFa, IL- I@, and IL-6 serum levels are elevated ( 10- Statisfical analj~sis.Differences between groups were analyzed 14). with f test. Differences were considered significant at p < 0.05. The precise physiologic and, possibly, pathophysiologic role of All data are expressed as mean + SEM. systematically present cytokines is still unclear. High levels of Patients. During a 6-mo period, 18 consecutive newborns with cytokines might cause direct damage (2, 3, 15). For instance. clinical suspicion of sepsis were evaluated. Two or more of the injection of high amounts of recombinant human IL-l can following ciinical symptoms were considered to be indicative for sepsis (26.32.33). The symptoms used in the study were lethargy. Received March 16. 1992; accepted October 22. 1992. Correspondence: E. de Bont. Department of Pediatrics. Division of Neonatology, apneic spells, poor peripheral circulation. and poor feeding. The Academisch Ziekenhuis Groningen. Oostersingel 59. Groningen. The Netherlands. 18 newborns underwent venipuncture for leukocyte count (I/ Supported in part by Medgenix. Fleums. Belgium. L): leukocyte differentiation: measurement of C-reactive TNFn.. IL-13. AND IL-6 IN NEONATAL SEPSIS 38 1 (&nL or mg/L). TNFn. IL- I@. and IL-6 (pg/mL or ng/L): and group were found (p < 0.01) (Fig. 1 ). IL-10 plasma levels were blood culture. All patients began taking antibiotics at the time detectable in nine out of the 10 newborns with sepsis. Values of of suspicion of sepsis. 18 t 5 pg/mL (ng/L) versus 7 f 1 pg/mL (ng/L) in the control Sepsis was defined as clinical suspicion and a positive blood group were measured (p < 0.01) (Fig. 2). TNFn plasma levels culture, or as clinical suspicion and inflammatory phenomena were found to be elevated in eight of the 10 newborns with sepsis. within 72 h: leukopenia less than 5. 109/L. a leukocytosis greater Values were 560 2 234 pg/mL (ng/L) in the septic group versus than 20. 109/L with an increase of the total immature neutrophil 36 + 4 pg/mL (ng/L) in the control group (p< 0.01) (Fig. 3). In percentage, and a C-reactive protein greater than 50 &mL (mg/ one patient with sepsis due to Staph!~lococcus epidermidis, the L) were considered inflammatory phenomena. Sepsis was found TNFn value increased slightly during the first 12 h of therapy in 10 of 18 patients; of those 10, eight had positive blood cultures. [from 73 to 103 pg/mL (ng/L)]. With the improvement of the The other two sepsis patients (no. 8 and 10) had negative blood clinical situation after the start of therapy. TNFa, IL-18, and IL- cultures, probably because their blood was obtained only after 6 plasma levels decreased and normalized in all patients in nearly they started receiving antibiotic therapy. Their blood parameters 2 d (Figs. I. 2. and 3). (as defined above) indicated sepsis. In eight patients. sepsis was In the septic group, no correlation was found between the not indicated by blood culture or blood parameters; these patients rectal temperature at the time of the first blood sample and the were not included in the study. Antibiotics were discontinued in TNFn, [email protected] IL-6 plasma level in the first blood sample (Fig. these patients after 3 d, and they recovered within a few days. 4). The control group included 22 healthy controls (1 1 females. Although low. the initial IL-I8 level proved to be correlated 11 males; mean gestational age 36.5 f 1 wk; mean birth weight with the difference between the mean diastolic blood pressure 2867 + 178 g; mean age 4 + 1 d). Blood for measure- according to birth weight minus the measured diastolic blood ments was obtained once when blood samples were taken for pressure (r = 0.66. p = 0.04: 34) (Table I). The elevation of the glucose, bilirubin. or Hb measurements. initial TNFn plasma levels did not correlate significantly with In one indiv~dualin the control group, an IL-I@plasma level the difference in diastolic tension, nor did the IL-6 plasma levels. was not measured due to an inadequate volume of the plasma sample. One patient in the septic group was lost for followup due DISCUSSION to transfer to another hospital. In this case. cytokine plasma levels were not available on the 2nd and 3rd d. In four patients In this report. we show that newborns with gram-positive sepsis in the septic group, the C-reactive protein level was not measured have elevated TNFn and IL-6 plasma levels, whereas IL-18 is due to inadequate sample volume. Patient data of the 10 septic only slightly increased. The observed increases of TNFa and IL- newborns (mean gestational age 33 + 1 wk. mean birth weight 6 plasma levels in this study extend the results obtained in 2005 t 266 g) are shown in Table I. The antibiotic regimen was children and adults during gram-negative sepsis (10-14). The ampicillin ( 150 mg/kg/d) and cefotaxime ( I00 mg/kg/d). when increase of TNFa in this study extends the findings reported in newborns became septic in the first days of life (n = 5). In the a recent paper in which TNFn was measured only once in case of sepsis in newborns aged 7 d or more (n = 4). the initial newborns suspected for sepsis (31). antibiotic therapy was flucloxacillin (100 mg/kg/d) and cefotax- New in this study are the profiles of TNFa. IL-I@,and IL-6 ime (100 mg/kg/d). The rectal temperature was monitored every plasma levels during the first 61 h of sepsis. the correlation 3 h. Diastolic tension was monitored continuously with an between the IL- I @ plasma levels and the decrease in diastolic arterial catheter (n = 2) or every 15 min with an automatic tension. and the obvious lack of a febrile response during sepsis sphygmomanometric method (Dynamap, Criticon Inc.) (n = 8). in newborns with low IL- I p plasma levels. When the clinical situation deteriorated during treatment with Although in ritro studies observed a defective production of these antibiotics. therapy was changed to gentamicin (5 mg/kg/ IL-6 and TNFa by umbilical cord blood monocytes in cell d)and imipenem (50 mg/kg/d). This was the case in the neonate cultures. newborns can produce as much TNFa and IL-6 during with the Enterohacter sepsis only. None of the newborns died. sepsis with gram-positive bacteria as adults or children with sepsis (1 1-14. 35, 36). In our present in rivo study. the production of TNFa and IL-6 proved to be independent of the gestational age. RESULTS even though in ;3itrostudies observed dependence on the gesta- tional age (37-39). In all the newborns with sepsis. IL-6 plasma levels were ele- Animals and humans given an i.v. injection of recombinant vated at the time of the first blood sample. Values of 79 700 t human IL- I. lipopolysaccharide. or endotoxins developed de- 37 500 pg/mL (ng/L) rc7rsus55 28 pg/mL (ng/L) in the control creased systemic arterial pressure (6. 17- 19. 40. 4 1 ). Human Table 1. Clinical data ~fpatic~tgrolrp* CRP (pg/ TNFn IL-Id IL-6 Temp Blood Leuko mL or (pg/mL or (pg/mL or (pg/mL or Patient GA (wk) BW (g) Sex Age (d) ("C) BP (mm Hg) ABP culture (xlOq/L) i% mg/L) ng/L) ng/L) ng/L) 1 40.0 4850 F 2 37.9 39/26 19 GBS 4.9 8 387 327 38 89 700 2 35.0 1350 F 7 38.5 43/32 -4 S. cpid. 10.7 16 180 10 523 3 29.0 1320 F 15 38.3 55/34 -6 S. epid. 10.6 8 11 103 10 148 4 32.0 1440 M 8 38.0 64/53 -24 S. clpid. 17.5 1 11 45 8 3 1 5 32.5 1320 F 7 38.5 28/17 11 Enthac. 4.4 5 159 902 47 410 000 6 26.0 950 F 1 37.5 33/23 3 GBS 3.7 5 75 144 9 142 000 7 34.0 2320 M 1 37.7 47/28 5 GBS 2.0 4 30 13 66 300 8 31.0 1706 M 7 38.2 35/29 1 2.3 5 71 2200 32 55 500 9 37.0 2980 F 1 36.8 92/55 -19 GBS 2.7 21 25 6 30 400 10 34.0 1830 F 1 37.5 40125 6 1.5 0 1640 4 2 300 * GA. gestational age: BW. birth weight: age. age at the moment of diagnosis: temp. temperature at the moment of diagnosis: BP. blood pressure at the moment of diagnosis: ABP. mean blood pressure according to birth weight minus measured blood pressure at the time of diagnosis: leuko. leukocyte count: i%. immature neutrophil percentage: CRP. peak values of C-reactive protein within 72 h (pg/mL or mg/L): GBS. group B streptococcus: S. epid.. Staph?~lococc~c.reprdermidis: Entbac. Enterobacter. TNFa. IL-I B. and IL-6 are levels in plasma. 382 DE BONT L-T .-IL.

0 10-14 1~~303~~42 51:61 contr t~me(hours 1 Fig. I. IL-6 plasma concentrations during the first 6 1 h of treatment in 10 newborns with sepsis. IL-6 plasma levels as mean k SEM. in ng/ mL or rg/L (total number of neonates included in each group of hours is indicated abokc ;hc column). Fig. I. IL- I.! pl-sm:! concentrations I.? the rectal temperature. IL- 16 plasma level (pg/mL or ng/L) of each newborn in the patient group (0) and the control group (e)in the first blood sample plotted against the rectal temperature ('C) of every individual newborn at the time of the first blood sample (R= 0.378. p = 0.28). are remarkably low in our study, IL-I@plasma levels measured it1 ~ivoduring sepsis in newborns appear to be correlated with the decrease in diastolic tension according to birth weight. Before conclusions can be drawn. however. this needs further investi- gation. IL- I@ has been indicated as the most pyrogenic cytokine ( 16). Systemic IL-I@induces a sudden prostaglandin E? increase in the anterior hypothalamic center. This increase of hypothalamic time Ihourrl prostaglandin E? raises the set-point temperature. which results Fig. 2. 1L-I/3 plasma concentrations during the first 61 h of treatment in a febrile response ( 16). in 10 newborns with sepsis. IL-113 plasma levels as mean + SEM. in pg/ In contrast to adults and children. newborns have little or no mL or ng/L (total number of neonates included in each group of hours febrile response to infection or sepsis (32). There is no difference is indicated above the column). between the IL- Id secretion of stimulated cord blood monocvtes from preterm and term newborns and the secretion ofstimulated TNF a(pg/ml) monocytes of adults (37). However. IL-I@production by stimu- 1 lated cord blood monocytes was decreased in infants with infec- tious complications postpartum (38). In our study. the IL-I@ plasma levels found in newborns during sepsis were remarkably low. especially when compared with findings during adult sepsis in which IL- I @ plasma levels varied between 120 and 1500 pg/ mL (ng/L) (I I. 14. 43). During sepsis in newborns. the mono- cytes may be inable to secrete more IL-I@. Luger rt al. (44) observed an association between fatal outcome of sepsis in adults and an inability of IL-I production in monocytes of those patients. Apart from IL- I. some authors found a relation between IL-6 and fever. but IL-6 is thought to be less pyrogenic than IL- I@ (45. 46). IL-6 release is induced by TNF and IL-1: therefore, the levels of IL-6 also may often correlate with the amount of o 10114 19~30 35-42 51:61 cdntr. fever. Althoueh a moderate febrile resDonse was seen in some time (hours) L, newborns with sepsis. we found no correlation between the 3. Fig. TNFa plasma concentrationsduring the first 61 h oftreatment temperature degree and the IL- I@ or IL-6 plasma levels. in 10 newborns with sepsis. TNFa plasma levels as mean SEM. in pg/ + In conclusion, during neonatal sepsis with mainly gram-posi- mL or ng/L (total number of neonates included in each group of hours is indicated above the column). tive bacteria, TNFa. IL- 18, and IL-6 plasma levels were increased 15-fold. 2-fold. and 1.500-fold. resoectivelv. recombinant TNF also induces decreased blood pressure in IL-I@plasma levels in neonatal sepsis correlate with the de- animals and humans, but in animal stud,es the decrease in mean crease in diastolic blood pressure according to birth weight. blood pressure was delayed (21, 23). After bacterial challenge. Although there is a slight difference between the IL-I@plasma TNFa induces an increase in IL-1 appearance that can be re- levels in the groups. there is no correlation between the temper- duced x;Jith antib=dies ?= T?JFa (42). HypGtensior? dce to ature degree and the IL-10 plasma level. Also. the IL-6 plasma polysaccharide or endotoxins can be blocked with recombinant level is not correlated with the temperature degree. Therefore, IL-1 antagonist (17. 18. 40). This suggests that the we hypothesize that a low IL-I@ plasma level may explain the hypotension in studies with TNF. endotoxins. or lipopolysaccha- lack of a febrile response and that the increase of IL-6 plasma ride is due to IL-1 ~nduction.Although the IL-I@plasma levels levels and fever are not directly related. ITS NEONAT41 SEPSIS 383

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