Masters Theses

Date of Award

5-1995

Degree Type

Thesis

Degree Name

Master of Science

Major

Biomedical Engineering

Committee Members

Raymond A. Popp

Abstract

Research was initiated to investigate several immunological parameters of the transgenic sickle cell mouse strain, MHOAH-Tg58RuPp (Mouse with High Oxygen Affinity Hemoglobin-Transgenic #58 Rubin Popp), in order to advance the use of these mice as a model for human sickle cell disease. Humans suffering from sickle cell disease have an increased susceptibility to pyogenic bacterial infections which suggests a possible immune dysfunction. A simple procedure, the hemolytic plaque assay was used to compare the number of antibody secreting cells in spleens of MHOAH-Tg58RuPp, MHOAH and C57BL/6J mice. Both primary and secondary responses to sheep red blood cells were evaluated. An elevated number of IgM secreting ceUs, in primary and secondary responses, was detected in MHOAH-Tg58RuPp mice relative to the two control strains. Fewer plaque-forming cells (PFCs) of the IgG class in the primary response were found in the sickle cell mice than in either C57BL/6J or MHOAH mice. However, in the secondary response the IgG PFCs were much higher in number in the sickle cell mice relative to the controls. Therefore, a study was initiated to delineate the numbers of cells present in various leukocyte subsets in the spleens and peripheral blood of sickle cell and control mice. Leukocyte subsets were identified by flow cytometric and immunofiuorescence methodologies. Antibodies were used to detect cell surface antigens present on the separate leukocyte subsets. Increased numbers of macrophages (MACs) and lymphocytes were found in the spleen of sickle cell mice relative to both C57BL/6J and MHOAH mice. The increase in the number of lymphocytes was largely due to the increase in B cells and natural killer cells (NK). The T cells in MHOAH-Tg58RuPp were increased but not to the same extent as B cells. The T cell subsets, CD8+ and CD4+ cells, were increased differentially. The helper T cell subset (CD4+) was statistically significantly increased while the cytotoxic T cell subset (CD8+) was not significantly increased. The polymorphonuclear neutrophils (PMNs) were statistically significantly increased. The largest cellular increases in the leukocyte subsets measured occurred in the MAC and NK populations. An increase in MACs could be explained, in part, by the increased use of MACs to remove the misshapen erythrocytes in the sickle cell mice. Studies involving sickle cell patients use peripheral blood mononuclear cells rather than spleen cells, so a comparative study of peripheral blood and spleen leukocyte subsets of the sickle cell mouse was conducted. Examinations of the peripheral blood subsets revealed results similar to those revealed by the splenic examinations. B cells and T cells (including CD4+ and CD8+) subsets were increased in number. While the NK cells, MACS, and PMNs of the spleen were increased in number compared to controls, there was a decrease in the number of these in peripheral blood subsets. The number of NK cells found in peripheral blood from MHOAH mice is less than half that in sickle cell mice and C57BL/6J mice. Increased numbers of splenic NK cells were found in MHOAH and sickle cell mice (peripheral blood NK cells: MHOAH< sickle cell< C57BL/6J, splenic NK cells: C57BL/6J≤ MHOAH

Results from these studies suggest that perturbations in the CD8+ T cell subsets in sickle cell mice may be caused by physiological conditions associated with the high oxygen affinity hemoglobin in both MHO AH and sickle cell mice, Perturbations in the B cell and CD4+ subsets appear to be associated with the increase in hematopoiesis and splenic cellularity. Perturbations in the NK, PMN and MAC subsets in the sickle cell mice may result from the production and polymerization of HbS Antilles, red cell sickling, and the pathobiology in several organs of the sickle cell mice. These results show that perturbations exist in MHOAH-Tg58RuPp mice. Nevertheless, the perturbations in the leukocyte subset populations did not cause a deficiency in the immunological functions examined in these transgenic sickle cell mice.

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