A Novel Role of CD40/CD40Ligand Dyad in Regulation of Bone Marrow Granulopoiesis.

Abstract 4556

Members of the tumor necrosis factor / tumor necrosis factor receptor (TNF/TNFR) superfamily are involved in bone marrow (BM) homeostasis by regulating the survival, apoptosis, proliferation, and differentiation of hematopoietic progenitor and precursor cells. The CD40 Ligand (CD40L)/CD40 molecules belong to the TNF/TNFR superfamily; we have recently showed that the CD40/CD40L interaction on BM CD34⁺ cells accelerates the apoptotic cell death. The aim of the present study is to investigate the distribution and function of the CD40/CD40L molecules in the BM granulocytic progenitor and precursor cell populations as well as the effect of their interactions on the stromal release of cytokines related to granulopoiesis. BM samples were obtained from posterior iliac crest aspirates from 19 hematologically healthy subjects after informed consent. We evaluated: (a) the surface expression of CD40 and CD40L on immunomagnetically sorted CD34⁺, CD34^-/CD33⁺, CD34^-/CD33^-/CD15⁺ cells representing sequential stages of the granulocytic development, using flow cytometry in steady state conditions and following 20-hour incubation with 25ng/ml recombinant-human (rh) TNFα; (b) the proportion of apoptotic cells and the level of caspase-3 activation following CD40 engagement in the above cell populations using flow cytometry and a chromo-enzymatic method, respectively; (c) the production of granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) by long-term BM culture (LTBMC) stromal cells upon induction with rhCD40L (1μg/ml), using an enzyme-linked immunoabsorbent assay (ELISA) in culture supernatants. We found that the basal expression of CD40 and CD40L on the CD34⁺, CD34^-/CD33⁺, CD34^-/CD33^-/CD15⁺ cells was 3.34%±3.90% and 1.8%±1.03%, 16.74%±14.69% and 6.96%±4.68%, 3.22%±3.36% and 2.26%±2.71%, respectively. Following incubation with rhTNFα a substantial increase was obtained in the expression of CD40 in all the above cell populations compared to baseline (37.18%±12.51%, 29.25%±14.81%, 15.84%±6.28%, respectively) (p<0.0001, p=0.0003, p=0.0002 respectively). Upon engagement of the TNF-induced CD40 in the CD34⁺, CD34^-/CD33⁺, CD34^-/CD33^-/CD15⁺ cells, a significant increase was obtained in the proportion of apoptotic cells (13.98%±10.43%, 27.24%±19.30%, 25.64%±14.82%, respectively) in comparison to TNF-induced cells without CD40 ligation (5.25%±4.04%, 11.6%±9.04%, 12.42%±5.22%, respectively) (p=0.0071, p=0.0296, p=0.0169, respectively). In keeping with this finding was the significant increase in the caspase-3 activation in all the above populations upon ligation of CD40 compared to baseline (p<0.0001, p=0.0281, p=0.0046, respectively). CD40 was expressed in both CD45⁺ (8.25%±8.31%) and CD45^- (8.46%±4.93%) adherent cell populations (hematopoietic and non hematopoietic, respectively) of confluent LTBMCs (week-3). Following 48-hour incubation of confluent LTBMC stromal layers with rhCD40L, a significant increase was obtained in the production of G-CSF and GM-CSF (2171±2161 pg/ml and 84.10±84.25 pg/ml, respectively) compared to unstimulated cultures (885±1099 pg/ml and 26.57±28.25 pg/ml, respectively) (p=0.0041 and p=0.0074, respectively). These data suggest that the CD40/CD40L interactions, although minimal under steady state conditions, display a dual role in regulation of granulopoiesis under inflammatory conditions by affecting both the BM granylocytic progenitor/precursor cells and the BM microenvironmental cells. More specifically, the expression of CD40 is upregulated in all stages of granulocytic development under the influence of TNFα and upon ligation with CD40L results in acceleration of the apoptotic cell death. The induction of G-CSF and GM-CSF production by BM stroma may represent a compensatory mechanism to rescue granulocytic progenitor/precursor cells from apoptosis. These mechanisms may have a role in the pathogenesis of chronic idiopathic neutropenia where there is evidence for both increased BM TNFα and serum CD40L levels.