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Abnormal response to granulocyte colony-stimulating factor (G-CSF) in canine cyclic hematopoiesis is not caused by
B R Avalos1, V C Broudy, S K Ceselski
1Department of Internal Medicine, Ohio State University, Columbus 43210.
Insights
Cyclic hematopoiesis in grey collies shows reduced neutrophil precursor response to granulocyte colony-stimulating factor (G-CSF). This defect is not due to G-CSF receptor issues but a downstream signaling problem.
Area of Science:
- Hematology
- Cell Biology
- Molecular Biology
Background:
- Cyclic hematopoiesis is linked to decreased responsiveness to granulocyte colony-stimulating factor (G-CSF).
- The canine model of cyclic neutropenia (grey collies) provides a valuable system to study this disorder.
- Understanding the cellular basis of G-CSF unresponsiveness is crucial for deciphering cyclic hematopoiesis pathophysiology.
Purpose of the Study:
- To investigate if the abnormal G-CSF response in canine cyclic hematopoiesis originates at the progenitor cell level.
- To determine if defective G-CSF receptor expression causes the observed G-CSF unresponsiveness in grey collies.
- To explore the G-CSF signal transduction pathway in neutrophils from normal and grey collie dogs.
Main Methods:
- Assessing neutrophil precursor response to G-CSF in vitro using bone marrow mononuclear cells.
- Quantifying G-CSF receptor expression and binding affinity on neutrophils via radioligand binding and Scatchard analysis.
- Identifying G-CSF binding protein size using chemical cross-linking.
- Analyzing G-CSF receptor mRNA transcripts via Northern blot.
- Investigating early G-CSF signal transduction events by measuring tyrosine phosphorylation of p80.
Main Results:
- Neutrophil precursors from grey collies required significantly higher G-CSF concentrations for colony growth compared to normal dogs.
- G-CSF receptor number, binding affinity, and protein size were similar in neutrophils from both normal and grey collie dogs.
- No detectable abnormalities in G-CSF receptor mRNA were found in grey collie neutrophils.
- G-CSF treatment induced comparable tyrosine phosphorylation of an 80-kD protein (canine c-rel) in both normal and grey collie neutrophils.
Conclusions:
- The reduced responsiveness to G-CSF in canine cyclic hematopoiesis resides in neutrophil precursors.
- The defect is not attributable to alterations in G-CSF receptor expression, binding, or size.
- The findings suggest a defect in the G-CSF signal transduction pathway distal to receptor binding, not involving early p80 tyrosine phosphorylation.
Abstract:
A decrease in responsiveness to granulocyte colony-stimulating factor (G-CSF) has been implicated in the pathophysiology of cyclic hematopoiesis. Using the canine model of cyclic neutropenia, we examined the response of neutrophil precursors to G-CSF in vitro and G-CSF receptor expression in neutrophils from grey collie dogs to determine whether the abnormal response observed to G-CSF in vivo in this disorder is present at the level of the progenitor cell and is caused by defective G-CSF receptor expression. Bone marrow mononuclear cells from grey collie dogs required sevenfold higher G-CSF concentrations than normal dog cells to achieve half-maximal colony growth [56 pmol/L v 8 pmol/L). Receptor binding assays with 125I-labeled G-CSF and Scatchard analyses of the equilibrium binding data were consistent with expression of a single class of high-affinity receptors for G-CSF on neutrophils from both normal dogs and grey collies with similar receptor numbers (56 to 446 sites/cell v 78 to 199 sites/cell) and binding affinities (28 to 206 pmol/L v 84 to 195 pmol/L). Chemical cross-linking studies identified a G-CSF binding protein of approximately 120 kD on neutrophils from grey collies, similar in size to that on normal dog neutrophils. No abnormal G-CSF receptor mRNA transcripts were detected in neutrophils from grey collie dogs by Northern blot analysis. Treatment of both normal and grey collie neutrophils with G-CSF rapidly induced tyrosine phosphorylation of an 80-kD protein that behaved like canine c-rel. These results demonstrate that the abnormal responsiveness to G-CSF in canine cyclic hematopoiesis is present in neutrophil precursors and is not associated with demonstrable alterations in the number, binding affinity, or overall size of the G-CSF receptor in neutrophils, or with defective tyrosine phosphorylation of p80. These data suggest that cyclic hematopoiesis is caused by a defect in the G-CSF signal transduction pathway at a point distal to G-CSF receptor binding that does not involve the early biochemical events leading to p80 tyrosine phosphorylation.