Leukocyte Integrin CD18 Expression Mediates Transient Neutropenia Following G-CSF Administration.

Abstract 3587

Poster Board III-524

The mechanism whereby neutrophils traffic from the circulation in response to G-CSF has remained unclear despite the observation of ourselves and others that there is a dramatic, yet transient, loss of circulating neutrophils shortly following the administration of G-CSF in humans, non-human primates, and mice (Gordon BC, et al. Exp Hematol. 35:872-8, 2007). To determine the role of the CD18 leukocyte integrin on neutrophils in the egress of neutrophils from the circulation, we used dogs with canine leukocyte adhesion deficiency (CLAD), a genetic disease in which a mutation in CD18 prevents CD18 surface expression. We selected CLAD dogs who had 5-10% CD18+ neutrophils following either matched littermate allogeneic transplant or autologous gene therapy for CLAD. Three CLAD dogs meeting these criteria were evaluated. Three carrier dogs served as controls. G-CSF was administered at 10μg/kg SQ to all six animals. Peripheral blood samples (EDTA) were taken immediately prior to G-CSF administration, and at 15, 30, 60, 120, 240 minutes, and 24 hours following G-CSF administration. Total white blood cell counts, neutrophil counts, and the number and percentage of CD18+ peripheral blood leukocytes were assessed. As anticipated, the control dogs had a 60% decrease in circulating neutrophils 30 minutes following G-CSF administration: the mean +/− standard of deviation (SD) absolute neutrophil baseline count decreased from 6806+/−1072/μL to 2727+/−767/μL. In five control animals the neutrophil nadir occurred at 30 minutes post-G-CSF, and in one control dogs it occurred 15 minutes following G-CSF administration. Experimental CLAD dogs had only a 35% decline in neutrophil numbers at 30 minutes, from a mean baseline of 6777+/− 672/μL to 4433+/−265/μL. In these dogs the neutrophil count returned to pre-G-CSF levels by 60 minutes post-G-CSF. By 24 hours after G-CSF, the neutrophil level was increased 3-fold from baseline. Immunophenotyping using an anti-CD18 and a canine specific anti-neutrophil PE conjugated antibody indicated that only the CD18+ neutrophils disappeared from the circulation following G-CSF administration. At baseline the transplanted CLAD dogs had a mean of 15.2+/−3.9% CD18+ peripheral blood leukocytes, of these 50.7+/−7.1% were CD18+ neutrophils. Thirty minutes following G-CSF administration the mean+/−SD percentage of CD18+ leukocytes declined to 13.7+/−3.7% with 33.4+/−5.8% being neutrophils. There was also a slight decline in CD14+CD18+ monocytes from 6.2 +/− 1.5% to 4.0 +/− 1.2%, which was not observed in the controls. There was no change in CD18- leukocyte numbers. The percentage of CD18+ neutrophils returned to baseline by 60 minutes and remained there at subsequent time points. These results demonstrate that the CD18 leukocyte integrin on circulating neutrophils mediates the transient neutropenia associated with G-CSF administration.