Activation of protein tyrosine kinase: a possible requirement for fixed-bacteria and lipopolysaccharide-induced increase in human natural killer cell activity.
Preincubation of peripheral blood lymphocytes (PBL) from drug-free, healthy volunteers with either the protein tyrosine kinase inhibitor genistein (GNT, n = 10, final concentration 200 microM) or the protein kinase A activator dybutiryl-cyclic-AMP (cAMP, n = 11, final concentration 10 microM), resulted in a significant inhibition of natural killer cell activity (NKCA, expressed as percentage of specific chromium release). With the exception of 4 out of the 11 cAMP-treated samples, individual values for NKCA in the drug preincubated specimens were at least 20% below the same subject baseline activity; furthermore, NKC lytic function was non-detectable in 4 out of the 10 and in 1 out of the 11 samples pretreated with either GNT or cAMP, respectively. PBL preincubation with glutaraldehyde-fixed Gram-negative bacteria (GNB, n = 13, final GNB-to-effector cell ratio of 50 : 1) resulted in a statistically significant increase in NKCA (baseline (x +/- SD) of 21.6 +/- 16.4 and bacteria treated samples of 41.5 +/- 24.6, respectively, Student's paired t-test p < 0.05). At least a 20% increase in NKC lytic function over its own baseline value was recorded for 11 out of the 13 samples tested (Table 1). Preincubation with GNB and GNT (5 samples) not only blocked the immunostimulant effects of GNB (Student's paired t-test p < 0.05), but in most cases individual values for NKCA were similar to those recorded for GNT-only treated samples. Use of cAMP instead of GNT also blocked, but to a smaller extent, the GNB-produced increases in NKC lytic function (paired Student's t-test < 0.05). PBL preincubation with lipopolysaccharide (LPS, n = 11, final concentration 50 micrograms/ml) resulted in a statistically significant increase in NKCA (baseline (x +/- SD) of 20.7 +/- 14.1 and LPS treated samples of 39.2 +/- 18.5, respectively, Student's paired t-test < 0.05). At least a 20% increase in NKCA over its own baseline value was observed for each and everyone of the 11 samples studied (Table 2). Addition of LPS and GNT to the incubation mixture resulted not only in inhibition of the NKCA upmodulating LPS effects (Student's paired t-test p < 0.05), but each and everyone of the individual samples' NKCA were, in fact, significantly lower than their corresponding control baseline values and similar to those recorded for GNT-only treated samples. However, the use of LPS and cAMP (Table 2) produced less dramatic results, significant inhibition of LPS effect were recorded in only 2 samples (Nos 8 and 10), and individual NKCA in the remaining 3 specimens was significantly higher than the corresponding baseline value. Whereas experimental results obtained with GNT support the involvement of PTK-dependent pathways in the stimulation of human NKCA produced by GNB and LPS, cAMP experiments suggest modulation of PKA-dependent pathways as responsible for the decrease in NK lytic function produced by a number of chemicals involved in the pathophysiology associated with certain forms of stress, including septic shock. Further research in this area could help in the rational design of pharmacological approaches for the treatment of these conditions.
Puente
J
J
Department of Biochemistry and Molecular Biology, Faculty of Chemical and Pharmaceutical Sciences, Santiago, Chile.
Salas
M A
MA
Canon
C
C
Miranda
D
D
Wolf
M E
ME
Mosnaim
A D
AD
D000328:Adult; D001419:Bacteria / Q000737:chemistry*; D001427:Bacterial Toxins / Q000494:pharmacology; D003994:Bucladesine / Q000494:pharmacology; D004768:Enterotoxins / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D005260:Female; D019833:Genistein; D006801:Humans; D007529:Isoflavones / Q000494:pharmacology; D007694:Killer Cells, Natural / Q000502:physiology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D008875:Middle Aged; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000737:chemistry
D001427:Bacterial Toxins; D004768:Enterotoxins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D007529:Isoflavones; D008070:Lipopolysaccharides; D003994:Bucladesine; C027538:heat-labile enterotoxin, E coli; D019833:Genistein; D011505:Protein-Tyrosine Kinases