Pediat Res 11 - PDF document

1 Pediat. Res. 11: 677-()1\0 (1977) Chem
Pediat. Res. 11: 677-()1\0 (1977) Chemotaxis complement monocytes neutrophil newborn phagocytes Cellular and Humoral Components of Monocyte and Neutrophil Chen1otaxis in Cord Blood SAYITA G. PAHWA.""'' RAJENDRA PAHWA. ELENA GRII\IES. AND ELIZA13ETII SI\IITII\VICK Departlllt'lll of Pediatrics aml/nmrwwlogy, Memorial Sloa/1-1\ellcrillg Ca11ca Centa, Nell' York, New York, USA Summary 1\lonoqte and polymorphonuclear neutrophil �(JI\IN) chemo­ taxis was studied in cord blood from healthv term infants. 1\Jono­ c;yte chemotaxis was normal to increased ( 115-126%) whereas PI\IN chemotaxis was decreased (79%) in comparison with that of healthy adult l'ontrol subjects. Generation of chemotactic factors from cord sera was impaired, being 55% of that ated by J)(Wied normal human serum (I'NIIS). Cord serum was less inhibitory than pooled adult human serum fur adult mono­ qtes when the cells were suspended in HI% serum and tested for chemotaxis. No inhibition of chemotactic factors by either cord or adult sera was observed. The dissociation of chemotactic response of the two diiTerent phagocytic cells may represent a protecth·e mechanism whereby one cell can compensate for a defect in the res1lonse of the other. Speculation Simultaneous anal_ysis of monocyte and neutrophil chemotaxis may reveal a defect restricted to one cell type. The human newborn infant is uniquely susceptible to infection with a variety of

2 infectious agents (bacterial, viral.
infectious agents (bacterial, viral. fungal, and protozoal) which often results in disseminated disease. Many studies of the defense been done, including studies of cell-mediated and humoral immunity ( 12, II-i), neutrophil function (lJ, 20. 23). inflammatory response (R, 10), and the complement system (2, 17). Little information is available about the monocyte, a cell of great importance in both the afferent and efferent limb of the immune response. Evidence in mice (I), rats (3), anti rabbits (2-1) suggests that the neonate has a deficiency in macrophage function. In humans, evaluation of the newborn infant's inflammatory response using the Rebuck skin window showed a ddaycd and diminished accumulation of monocytcs (8), suggesting a possible defect in monocyte migra­ tion. Two recent studies on mon conflict­ ing results. One, done in 1-to 5-day-old infants, reports de­ creased chemotaxis ( 15) and the other, done in cord blood, reports normal chemotaxis (16). The investigations reported here were designed to cvalualc the cellular and humoral components of monocyte chemotaxis in the newborn infant, using cord blooLI. Previously, Miller (21) had reported an impairment of neutrophil chemotaxis and in the generation uf serum chemotactic factors in 3-to 5-day-old infants. The present study was expanded to include neutrophil chemotaxis (I) to sec whether the defects described in newborn infants were also present in corLI blood, an

3 LI (2) to sec whether the responses o
LI (2) to sec whether the responses of monocytcs and ncutrophils resembled each other. MATERIALS AND METHODS Heparinized (20 units/ml) cord blood, obtained from experiment, blood from a healthy adult was tested simultane­ously. ISOLATION OF CELLS Mononuclear leukocytes were isolated by density gradient centrifugation on a sodium mctrizoatc-Ficoll solution (Lympho­prep. Nyegard and Co., Oslo) (5). The cells were washed three times and resuspended in RPI\11 (Gibco) supplemented with penicillin 50 units, streptomycin 50 Jlg. and glutamine 2 ml\1/ml. As simultaneous analysis of monocytcs by myelopcroxidasc stain and Wright stain were in close agreement. the percentage of monocytcs was routinely determined by a myelopcroxidasc stain ( 13 ); Wright stain was done to exclude contamination by granu­ locytes. The final cell concentration was adjusted to I I on monocytcs/ml. To isolate polymorphonuclear ncutrophils, the granulocyte­ rich. red blood cell sediment from the Lymphoprep gradient was resuspended in RPM I I ()40; the red blood cells were then scdimentcd with 6% dextran in saline solution (Abbott). RcLI 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 (MEM, Gibco) at a concentration of 1.0 x 10'; PMN's/ml. CIIEt\tOTACTIC STI1\IULI Lymplzocytc-dcril'cd Clzmzolllctic Factor ( LDCF). This l

4 ym­ phokinc was prepared by concanaval
ym­ phokinc was prepared by concanavalin A (I 0 1-lg/ml) stimulation of lymphocytes. Cultures containing 2 X I on lymphocytcs/ml RPMI 1640 without serum were incubated at 37o in 5% SI5% humiLiificd air for 48 hr. After centrifugation, the superna­tant Ouid was stored in aliquots at -70° until usctl. LDCF was used as the chemotactic stimulus fur monocytes in the following proportion: 30% LDCF, 40% RPMI 1640, and 30% Hank's balanced salt solution with Ca ++, Mg+ +,and I '.:o gelatin (!lOSS- G). Actimtcd serum (AS). Pooled normal human serum (PNHS) was stored in aliquots at -700 and thawed just before usc. The serum was activated with endotoxin (Esclzcriclzia coli 026:136, Difco), 300 1-lg/ml at 37° for 60 min, followed by inactivation at 56° for 30 used were serum 0.1 ml, endotoxin 0.1 ml, HBSS-G 1.0 ml, and MEM 0.8 ml, which gave a final serum concentration of 5%. This stimulus was tested against both monocytcs and PMN's. Appropriate controls, without any chemotactic stimuli, were tested simultaneously. These consisted of 40% medium (RPM I for monocytcs and MEM for PMN's) and 60% HBSS-G. CHEMOTAXIS ASSAY The test procedure was a modification of the method of Snyderman c/ at. (26). All assays were done in triplicate. Modified Boyden chemotactic chambers (Blind-Well, Ncuro­probc) were used. The wells of the chambers were filled with 0.2 ml of the chemotactic stimulus, delivered with a calibrated pi- 6