Effect of Hydroxyurea on Immunity in Sickle Cell Disease

Solo Kuvibidila, PhD, Principal Investigator
James Hempe, PhD
Research Associate
Biostatistician

Abstract
Sickle cell disease (SCD) affects 1 in 400 African-American newborns, with a career rate of 1 in 12. This disease is characterized by vaso-occlusive episodes, progressive multiple organ damage (spleen, heart, kidneys), and increased incidence of pneumococcal infection during childhood due to impaired immunity. Except for bone marrow transplantation, it has no cure. Hydroxyurea (HU) has been shown to be effective in reducing the frequency of pain crisis in children and adults. This chemotherapeutic drug, however, induces cytopenia. Its long term effects on immune responses has not been previously investigated. Considering its inhibitory effect on ribonucleotide reductase, the enzyme required for the biosynthesis of deoxyribonucleotides, we hypothesize that its long term use will interfere with in vivo T cell proliferation, maturation, and functions. We propose to investigate these potential effects in sickle cell mice that show abnormalities similar to those seen in humans. Our aims are to determine whether HU administration to 2-3 month old sickle mice for 1, 3, and 6 months alters:

A) the percentages of thymocytes, blood and spleen cells that express markers associated with T cell maturation: CD4+CD8+, CD4-CD8-, CD4+CD8-, CD4-CD8+

B) in vivo secretion of interferon-gamma (IFN-gamma), pro-inflammatory (tumor necrosis factor-", interleukin [IL]-1, IL6, IL8), and anti-inflammatory (IL10) cytokines in lipopolysaccharide-treated mice;

C) in vitro proliferative responses of spleen cells to mitogens, and secretion of TH1 (IFN-gamma, IL2) and TH2 (IL-4) cytokines;

D) in vitro cytotoxicity of spleen and peritoneal cells toward allogenic tumor cells. Sickle and wild type (C57BL/6) mice will receive either 0, 10, or 30 µg HU/g body weight by intraperitoneal injection, 5X/week (doses similar to those used in SCD patients). Cytotoxicity will be studied by chromium release from mastocytoma (p815) cells in mice primed 10 and 5 days earlier. The following techniques will be used in various experiments: flow cytometry for T cell subsets, ELISA for cytokines, coulter counter for hematologic indices, capillary electrophoresis for hemoglobin S and F analysis and 3H-thymidine uptake for cell proliferation. The proposed project will provide important information on whether HU may further compromise immunity in sickle cell disease. Data will allow us to plan for a study in SCD children.

Sponsor: NIH
Duration: 07/03 - 06/05