Lymphocytes Are Dispensable In Neutrophil Homeostasis.
Authors: Bugl Stefanie , Wiesner Tina , Kanz Lothar , Kopp Hans-Georg , Wirths Stefan 
Published: October 2019

Abstract
Abstract 2613

Introduction:
In contrast to red cell or platelet homeostasis, the feedback mechanisms involved in the control of peripheral neutrophil numbers are incompletely understood. Granulocyte-Colony Stimulating Factor (G-CSF) is generally accepted to be the most important determinant of neutrophil production and release from the bone marrow. Recently, a feedback loop including a specialized subset of T-lymphocytes (Tn cells) has been established to explain the correlation between peripheral neutrophil clearance and increased G-CSF production.

Methods:
Wild type C57bl/6 mice or NODSCIDcγ−/− received anti-Gr1 or anti-1A8 antibodies to deplete neutrophils. Hematopoietic tissues and peripheral blood were harvested at different times and analyzed on cellular, protein and RNA level.

Results:
Both anti-Gr1 and 1A8 antibodies reduced neutrophils effectively and persistently in vivo. The reaction on neutrophil depletion in the marrow uniformely consisted of an absolute increase in lin-/Sca1+/c-kit+ (LSK) cells and a shift of myeloid progenitors towards granulocyte-macrophage precursors (GMP) and common myeloid progenitors (CMP) at the expense of megakaryocyte-erythrocyte precursors (MEP). Of note, exogenous G-CSF resulted in identical changes. We therefore hypothesized that neutrophil depletion increases G-CSF through a feedback regulatory loop. Neutrophil depletion with anti-Gr1 antibody in wt mice increased G-CSF levels up to approximately 8-fold. While previous observations suggest that G-CSF may be passively regulated through receptor binding and internalization by mature neutrophils, we also found a 10-fold increase of G-CSF mRNA in the marrow. These findings are consistent with active feedback. Interestingly, the effects of 1A8 antibody on G-CSF were less pronounced. Instead, mice depleted of neutrophils with 1A8 antibody displayed predominant increases of M-CSF, suggesting redundant feedback pathways. Our results in wildtype mice treated with 1A8 antibody confirm previous data by Stark et al. (Immunity 2005; 22: 285–294), including increases of IL-23 and IL-17 after neutrophil depletion. However, when neutrophils were depleted in NODSCIDcγ−/− mice, who lack lymphocytes and NK-cells, both IL-23 and IL-17 remained unchanged, but G-CSF levels still increased markedly.

Conclusions:
Effective neutrophil depletion by antibodies directed against different neutrophil antigens uniformly results in major shifts in the hematopoietic bone marrow showing an increase in the number of LSK hematopoietic stem cells accompanied by a significant increase in myeloid progenitors at the expense of thrombopoietic, red cell, and lymphoid precursors. Our results demonstrate regulatory feedback loops of neutrophil granulopoiesis culminating in increased production of myelopoiesis stimulating cytokines such as G-CSF, GM-CSF, and M-CSF. The underlying chain of events includes IL-23 and IL-17 in wild type mice as previously described. However, additional redundant pathways exist in lymphocytopenic NODSCIDcγ−/− mice.

Disclosures:
No relevant conflicts of interest to declare.