ABSTRACT
Several cytokines are known to enhance FMLP-stimulated superoxide generation in human circulating blood neutrophils through binding to their specific receptors, a process referred to as the priming effect. The priming effects produced by recombinant human granulocyte CSF (rhGCSF) and TNF-alpha (rhTNF-alpha) on FMLP-stimulated superoxide production in human and rabbit blood neutrophils were compared with their effects in their respective tissue neutrophils, i.e., human salivary and rabbit peritoneal neutrophils. The receptor binding characteristics of rhGCSF and rhTNF-alpha were also compared between the two types of neutrophils. Both rhGCSF and rhTNF-alpha produced dose-dependent priming effects on FMLP-stimulated superoxide production in human blood neutrophils, whereas they failed to produce any priming effects in human salivary neutrophils. Similar results were obtained for the priming effects by rhGCSF in rabbit blood and peritoneal neutrophils. A decrease in receptor binding affinity, but not in receptor density, in tissue neutrophils was demonstrated by analyzing the binding of [125I]rhGCSF and [125I]rhTNF-alpha. These findings suggest that tissue neutrophils are less responsive to rhGCSF and rhTNF-alpha in the modulation of FMLP-stimulated superoxide generation. This is due at least in part to the lower affinities of GCSF and TNF-alpha to their receptors in tissue neutrophils. This marked difference in priming effects by cytokines between blood and tissue neutrophils may represent an early step in the defensive responses against invading microorganisms or Ags.
Subject(s)
Down-Regulation/drug effects , Granulocyte Colony-Stimulating Factor/pharmacology , Neutrophils/drug effects , Receptors, Granulocyte Colony-Stimulating Factor/biosynthesis , Receptors, Tumor Necrosis Factor/biosynthesis , Respiratory Burst/drug effects , Superoxides/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Animals , Blood Cells/drug effects , Blood Cells/metabolism , Exudates and Transudates/cytology , Filgrastim , Humans , Luminescent Measurements , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/metabolism , Organ Specificity , Peritoneal Cavity/cytology , Rabbits , Receptors, Granulocyte Colony-Stimulating Factor/genetics , Receptors, Tumor Necrosis Factor/genetics , Recombinant Proteins , Saliva/cytologyABSTRACT
The phosphate translocator protein of C(3) and C(4) mesophyll chloroplast envelopes was specifically labeled using the anion exchange inhibitor, 1,2-ditritio-1,2-(2,2' -disulfo-4,4' -diisothiocyano) diphenylethane ([(3)H](2)-DIDS). Intact mesophyll chloroplasts were isolated from the C(3) plants, Spinacia oleracea L. (spinach) and Pisum sativum L. (pea), and the C(4) plant, Zea mays L. (corn). Chloroplasts were incubated with 5 to 50 mum [(3)H](2)-DIDS and, in addition, pea chloroplasts were also incubated with pyridoxal phosphate/tritiated sodium borohydride. The chloroplasts were washed, the envelopes isolated and solubilized. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis, label from bound [(3)H](2)-DIDS was detected only in the 28- to 30-kilodalton protein (proposed C(3) phosphate translocator) for both C(3) and C(4) chloroplasts, as demonstrated by fluorography. In contrast, when pyridoxal phosphate/tritiated sodium borohydride was used to label pea chloroplasts, radioactivity was detected in several other bands in addition to the 29-kilodalton polypeptide. These findings suggest that DIDS is a much more specific inhibitor than reagents previously employed to study the phosphate translocator and could be used to isolate and characterize the differences in the C(3) and C(4) phosphate translocator protein(s).
