Human newborn polymorphonuclear neutrophils exhibit decreased levels of MyD88 and attenuated p38 phosphorylation in response to lipopolysaccharide.
OBJECTIVE
Human newborn infants have increased susceptibility to gram-negative bacterial infection. Since lipopolysaccharide (LPS) primes polymorphonuclear neutrophils (PMN) to enhance host defense functions, we investigated its effect on adult and newborn PMN in vitro.


METHODS
PMN were isolated from blood of healthy adults and umbilical cords of full term newborns using dextran and Ficoll-Paque gradient sedimentation. Gel electrophoresis and Western blotting of membranes were used to probe for Mitogen-Activated Protein (MAP) kinase p38 phosphorylation, Toll-like Receptor-4 (TLR-4) and Myeloid Differentiation Factor 88 (MyD88) on isolated PMN membranes using specific antibodies. LPS induced degranulation was assessed using CD66 expression on PMN measured by flow cytometry.


RESULTS
We show that p38 phosphorylation in newborn PMN is attenuated in response to LPS stimulation even though adult and newborn PMN have similar amounts of p38 protein. The degree of attenuation in newborn PMN is dependent on the osmolarity of the medium. In addition, LPS-induced degranulation, a process that is p38 dependent, was also absent in newborn PMN. Although the LPS receptor TLR-4 is present at similar levels on newborn and adult PMN, its downstream adaptor protein MyD88 was significantly diminished in newborn PMN compared to adult cells.


CONCLUSIONS
Although the mechanism of PMN priming by LPS is not fully understood, our results suggest that MyD88 and p38 phosphorylation are important pathways in the process and contribute to attenuated response of newborn PMN to LPS in vitro.
Al-Hertani
Walla
W
Department of Microbiology, Dalhousie University and IWK Health Centre, Halifax, Nova Scotia, Canada.
Yan
Sen Rong
SR
Byers
David M
DM
Bortolussi
Robert
R
D015703:Antigens, CD / Q000378:metabolism; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006801:Humans; D007231:Infant, Newborn; D008070:Lipopolysaccharides / Q000494:pharmacology*; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism*; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D015703:Antigens, CD; C069847:CD66 antigens; D015815:Cell Adhesion Molecules; D008070:Lipopolysaccharides; D053594:Myeloid Differentiation Factor 88; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D048051:p38 Mitogen-Activated Protein Kinases