Pediat. Res. 11: 677-()1\0 (1977) Chemotaxis neutrophil complement newborn monocytes phagocytes Cellular and Humoral Components of Monocyte and Neutrophil Chen1otaxis in Cord Blood SAYITA G . PAHWA.""'' RAJENDRA PAHWA. ELENA GRII\IES. AND E LIZA13 ETII SI\IITII\VICK Departlllt'lll of Pediatrics aml/nmrwwlogy, Memorial Sloa/1-1\ellcrillg Ca11ca Centa, Nell' York, New York , USA Summary experiment, blood from a healthy adult was tested simultane­ ously. 1\lonoqte and polymorphonuclear neutrophil (J>I\IN) chemo­ taxis was studied in cord blood from healthv term infants. 1\Jono­ ISOLATION OF CELLS c;yte chemotaxis was normal to increased ( 115-126%) whereas PI\IN chemotaxis was decreased (79%) in comparison with that Mononuclear leukocytes were isolated by density gradient of healthy adult l'ontrol subjects. Generation of chemotactic centrifugation on a sodium mctrizoatc-Ficoll solution (Lympho­ factors from cord sera was impaired, being 55% of that gener­ prep. Nyegard and Co., Oslo) (5). The cells were washed three ated by J)(Wied normal human serum (I'NIIS). Cord serum was times and resuspended in RPI\11 (Gibco) supplemented with less inhibitory than pooled adult human serum fur adult mono­ penicillin 50 units, streptomycin 50 Jlg . and glutamine 2 ml\1/ml. qtes when the cells were suspended in HI % serum and tested for As simultaneous analysis of monocytcs by myelopcroxidasc stain chemotaxis. No inhibition of chemotactic factors by either cord and Wright stain were in close agreement. the percentage of or adult sera was observed. The dissociation of chemotactic monocytcs was routinely determined by a myelopcroxidasc stain response of the two diiTerent phagocytic cells may represent a ( 13 ); Wright stain was done to exclude contamination by gra nu­ protecth·e mechanism whereby one cell can compensate for a locytes. The final cell concentration was adjusted to I .5 x I on defect in the res1lonse of the other. monocytcs/ml. To isolate polymorphonuclear ncutrophils, the granulocyte­ Speculation rich. red blood cell sediment from the Lymphoprep gradient was resuspended in RPM I I ()40 ; the red blood cells were then Simultaneous anal_ysis of monocyte and neutrophil chemotaxis scdimentcd with 6 % dextran in saline solution (Abbott). Rc LI may reveal a defect restricted to one cell type. blood cells remaining in the leukocyte-rich supernatant were lysed with hypotonic saline . The leukocytes were then washed three times and resuspended in minimum essential medium The human newborn infant is uniquely susceptible to infection (MEM , Gibco) at a concentration of 1.0 x 10'; PMN's/ ml. with a variety of infectious agents (bacterial, viral. fungal, and protozoal) which often results in disseminated disease. Many CII Et\tOTACTIC STI1\IULI studies of the defense mechanisms in newborn infants have been done, including studies of cell-mediated and humoral immunity Lymplzocytc-dcril'cd Clzmzolllctic Factor ( LDCF). This lym­ ( 12, II-i), neutrophil function (lJ, 20. 23). inflammatory response phokinc was prepared by concanavalin A (I 0 1-lg/ml) stimulation (R, 10), and the complement system (2, 17). Little information is of lymphocytes. Cultures containing 2 X I on lymphocytcs/ ml in available about the monocyte, a cell of great importance in both RPMI 1640 without serum we re incubated at 37o in 5 % the afferent and efferent limb of the immune response. Evidence SI5 % humiLiificd air for 48 hr. After centrifugation , the superna­ in mice (I), rats (3), anti rabbits (2-1) suggests that the neona te tant Ouid was stored in aliquots at -70° until usctl . LDCF was has a deficiency in macrophage function . In humans, e valuation used as the chemotactic stimulus fur monocytes in the following of the newborn infant's inflammatory response using the Rebuck proportion: 30% LDCF, 40% RPMI 1640, and 30% Hank's skin window showed a ddaycd and diminished accumulation of balanced salt solution with Ca ++ , Mg+ +,and I '.:o gelatin (!lOSS- monocytcs (8), suggesting a possible defect in monocyte migra­ G). tion. Two recent studies on monocyte chemotaxis give conflict­ Actimtcd serum (AS) . Pooled normal human serum (PNHS) ing results. One, done in 1- to 5-day-old infants, reports de­ was stored in aliquots at -700 and thawed just before usc . The creased chemotaxis ( 15) and the other, done in cord blood, serum was activated with endotoxin (Esclzcriclzia coli 026:136, reports normal chemotaxis (16). Difco), 300 1-lg/ml at 37° for 60 min, followed by inactivation at The investigations reported here were designed to cvalualc 56° for 30 min. The proportions used were serum 0 . 1 ml, the cellular and humoral components of monocyte chemotaxis in endotoxin 0.1 ml, HBSS-G 1.0 ml, and MEM 0.8 ml, which the newborn infant, using cord blooLI . Previously, Miller (21) gave a final serum concentration of 5 %. This stimulus was tested had repo rted an impairment of neutrophil chemotaxis and in against both monocytcs and PMN's. the generation uf serum chemotactic factors in 3- to 5-day-old Appropriate controls, without any chemotactic stimuli, were infants. The present study was expanded to include neutrophil tested simultaneously . These consisted of 40% medium (RPM I chemotaxis (I) to sec whether the defects described in newborn for monocytcs and MEM for PMN's) and 60% HBSS-G. infants were also present in corLI blood, anLI (2) to sec whether the responses of monocytcs and ncutrophils resembled each CH EMOTAXIS ASSAY other. The test procedure was a modification of the method of Snyderman c/ at. (26). All assays were done in triplicate. MATERIALS AND METHODS Modified Boyden chemotactic chambers (Blind-Well, Nc uro­ Heparinized (20 units/ml) cord blood, obtained from healthy probc) were used. The wells of the chambers were filled with 0.2 full term infants, was tested within 4 hr of delivery. In every ml of the chemotactic stimulus, delivered with a calibrated pi- 677 678 PAIIWA ET AL. pette (Finnpipette, Neuroprobe). Numbered filters (Nude­ eight showed a decreased response and three had a response pore) of pore size 5 J.Lm for monocytes and 3 J.LII1 for PMN's were equal to that of adult PMN's (Table 2). The mean chemotactic placed over the filled wells, taking care not to trap bubbles. The response of cord PMN's was 79% of that of adults (P < 0.05) filter retainers were then screwed in, and the upper compart­ I3ackground counts of cord and adult PMN's wen; similar. ments charged with 0.2 ml of the appropriate cell suspension. The chambers were incubated at 3 7o in humidified air for a GENERATION OF SERUM CHEMOTACTIC FACTOR period of 90 min for monocytes and 45 min for PMN's. After incubation, the upper compartments were aspirated and the Cord sera generated less chemotactic activity for PMN's than filters were removed and stained in Mayer's hematoxylin. Cells did PNHS. The chemotactic response of adult PMN's to acti­ migrating to the undersurface of the filter were counted, using vated cord sera was 55% of that seen with activated PNHS, P < an ocular grid (7 x 7 mm), with a magnification of 1 ,000 times 0.0005 (Table 3). for monocytes and 400 times for PMN's. Ten random grid fields were counted on every filter. The results were expressed as the SERUM INIIIBITORS OF CIIEI\IOTAXIS mean number of cells per ocular grid and as a percentage of the Adult mononuclear cells suspended in serum showed a lower normal. monocyte chemotactic response than that of cells suspended in medium alone. PNHS was more inhibitory than cord serum, STUDY OF SERUM FACTORS there being 51% inhibition of monocyte chemotaxis by PNIIS, Using the basic techniques described above, experiments were and 26% inhibition by cord serum (Table 4). Monocytes in cord done to evaluate the following. serum showed a consistently higher response to I 0% AS than Ability to Generate Chemotactic Factor. Cord sera and PNHS when suspended in PNIIS (P < 0.05). On the other hand, the were tested simultaneously for chemotactic activity after endo­ response of PMN's suspended in cord serum varied from a toxin activation, using adult PMN's as test cells. slightly decreased to a slightly increased response as compared Inhibitors of Cells. Normal monocytes and PMN's were sus­ with cells suspended in PNHS, but no significant differences pened in either 10% cord serum or 10% PNIIS; 10% activated were observed. Only minimal inhibition by either serum was normal serum was used as the chemotactic stimulus. seen. Cord cells similarly suspended in either PNHS or cord Jnhibitors/lnactivators of Chemotactic Stimuli. Cord serum or serum were not tested. normal serum, 5%, was added to 5% activated normal serum. This 10% serum mixture was used as the chemotactic stimulus SERUM INHIBITORS/INACTIVATORS OF CHEMOTACTIC FACTORS for normal PMN's. Similarly, cord or normal serum, 10%, was There was no evidence of any inhibitor or inactivator in added to the 30% LDCF and tested against normal monocytes. unfractionated cord or pooled normal serum to either of the chemotactic factors tested. Monocytes exhibited an increased RESULTS response to LDCF which had added cord or pooled normal human serum than when exposed to LDCF alone (Table 5). MONOCYTE CHEMOTAXIS Addition of either 5% cord or pooled adult serum to 5% The percentage of monocytes in cord mononuclear cells was consistently higher than that in adult cells: 25 ± 8% versus 16 ± Table 2. Response of cord and adult polymorphonuclear 5%, respectively. The final monocyte concentrations were ad­ neutroplzils ( PMN's) to endotoxin-activated normal serum (AS) justed as described above such that the chambers all received equal numbers of either cord or adult monocytes. LDCF was the Mean chemotactic response' chemotactic stimulus for 24 cord samples; AS was used as an Cord/ additional stimulus in 12 (Table I).