ABSTRACT
It has previously been reported that the biological activity of the human hematopoetic cytokine granulocyte colony stimulating factor (G-CSF) was reduced following incubation with human serum. The mechanism of action of serum has remained elusive although a number of possible mechanisms have been suggested including inactivation due to binding to the serum protein alpha(2)-macroglobulin (alpha(2)M) and degradation by serum proteases. The aim of this study was to clarify the conditions required by serum to reduce the biological activity of the cytokine and to define the mechanism involved. It has also been noted that G-CSF obtained from a CHO expression system (and therefore considered a glycosylated molecule) was resistant to serum inactivation unlike G-CSF obtained from an E. coli expression system (considered to be non-glycosylated). We used an enzymatic approach to remove the carbohydrate residues from glycosylated G-CSF and tested this material for its stability in serum. We additionally used a mutated G-CSF lacking glycosylation sites. We concluded that glycosylation was important in protecting against serum inactivation. We observed that serum reduced the biological activity of non-glycosylated G-CSF in a dose, and temperature dependent manner and deduced that the mechanism of action was dependent upon alpha(2)M bound serum protease enzymes.
Subject(s)
Carbohydrates/chemistry , Granulocyte Colony-Stimulating Factor/chemistry , Serum/metabolism , Animals , Biological Assay , CHO Cells , Cricetinae , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Glycosylation , Granulocyte Colony-Stimulating Factor/metabolism , Hydrogen-Ion Concentration , Temperature , Time Factors , alpha-Macroglobulins/metabolismABSTRACT
Recent clinical trials have shown that the new generation of acellular pertussis vaccines (Pa) can confer protection against whooping cough with negligible adverse reactions. We have compared the effects of pertussis whole cell and acellular vaccines on pulmonary immune responses after aerosol challenge in a murine model of infection. Mice were vaccinated with PBS, Pw or Pa and challenged with Bordetella pertussis by the aerosol route. Cytokine gene expression was analysed from lung tissue and cells; lung lymphocytes were re-stimulated in vitro and cytokines produced measured. The results obtained are consistent with the proposal that a strong Th-1 response is associated with bacterial clearance in both the non-vaccinated and Pw vaccinated mice. The acellular vaccine treated mice cleared the bacterial challenge (with an intermediate efficacy) in the presence of low levels of any of the cytokines assessed. This suggests that Pa protects via a Th-2 independent mechanism.