Inhibition of hematopoiesis in ltbmc is associated with nicotine induced down-regulation of cd44 expression

Bone marrow stromal cells (SC) are an integral component of the hematopoietic microenvironment and play a critical role in regulation of HSPC behavior. SC elaborate various positive and negative regulators as well as ECM and adhesion molecules that contribute in HSPC proliferation, differentiation and self-renewal. SC, as a target for extrinsic factors, may have a significant effect on hematopoiesis during both normal and pathological conditions. Exposure to pathophysiological factors could lead to an imbalance of SC functioning. Nicotine is a major component of tobacco smoke and its long-term exposure can result in hematological and immune imbalances. To examine if nicotine inhibits hematopoiesis by affecting the hematopoietic microenvironment, varying concentrations of nicotine were added to LTBMC from the first day of culture. Treatment of LTBMC with nicotine resulted in the inhibition of adherent layer formation in a dose dependent manner. Formation of a confluent adherent layer was significantly delayed in LTBMC treated with low doses of nicotine (105-10"'M), and dramatically affected at higher doses of nicotine (10^-3-10^-4M). Furthermore, we observed that in cultures treated with high doses of nicotine (10^-3-10^-4M), "cobble stone areas" were not formed. These areas were found to be smaller in cultures treated with low doses of nicotine (10^-5-10^-7M) compared to control cultures. Next, the influence of nicotine on hematopoiesis in LTBMC was examined. Administration of 10^-4M nicotine resulted in significant inhibition of non-adherent cell production during the entire period of culture. In addition treatment with nicotine resulted in a significant reduction in the numbers of myeloid progenitors in LTBMC. We next examined whether nicotine affects expression of adhesion molecules by SC as several of these cell surface receptors including CD44, o4, a2, β7 and VCAM-1 may participate in SC-HSPC interactions. MS-5 stromal cell line was used as model to study the effects of nicotine, as it supports growth of HSPC in vitro. Treatment with nicotine resulted in a 5-fold down-regulation of CD44 expression and 1.7-fold increase in β7 integrin expression. In contrast, the expression of other adhesion molecules remained unaffected by this treatment. We therefore conclude that exposure to nicotine leads to down regulation of CD44 expression, and an inhibition of initial adhesion of HSPC to SC resulting in significant reduction in HSPC numbers and reduced hematopoiesis in vitro.