Open Collections

Abstract

Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis, is a facultative intracellular pathogen capable of survival in macrophages. Several macrophage receptors including complement receptor type 3 (CR3, CDI lb/CD 18) are reportedly capable of binding MTB. It has been suggested that MTB survival in macrophages is determined by the receptor to which MTB binds before being phagocytosed. A CDI lb knockout mouse model was used as a source of macrophages to test whether CR3 is essential for the association of MTB with macrophages and to examine whether the absence of CR3 on macrophages alters the intracellular fate of MTB. Together, these research objectives were aimed at improving our understanding of the role CR3 plays in the pathogenesis of tuberculosis. Studies using both alveolar macrophages (AM) and peritoneal macrophages (PM) showed CR3 was not essential for MTB binding by macrophages, either in the presence or absence of serum. However, CR3 was involved in the association of MTB with macrophages in the absence of serum and was important for the efficient binding of MTB under conditions where serum was present. Compared to macrophages expressing CDI lb (CDI lb+/+), CDI lb-knockout (CDI lb-/-) macrophages were less able to associate with MTB, either opsonically or non-opsonically. Under opsonic conditions, the enhanced binding of MTB to macrophages was mediated by a heat-labile serum component, as heat-inactivation of serum abrogated the increased binding. Using immunoglobulin-deficient serum to opsonize MTB demonstrated that the increased binding of MTB was not mediated by immunoglobulins. The role of either the classical or alternative complement pathways in mediating the increased binding of MTB by PM was also investigated. The observations made here indicate that, in the presence of low serum concentrations, increased binding of MTB by CDI lb+/+ PM is mediated predominantly via activation of the classical pathway but is independent of immunoglobulins. The intracellular survival and replication of MTB following phagocytosis by either CDI lb-/- PM or CDI lb+/+ PM was investigated and no significant difference in the replication of MTB was detected where similar numbers of MTB were ingested. Therefore, the observations made here do not support the hypothesis that MTB exploits CR3 as a means to evade being killed by macrophages. As an alternative approach to the CDI lb knockout mouse model, an alveolar macrophage cell line, MH-S, was tested for its suitability as a model to investigate the role of CR3 in mediating the interaction of MTB with macrophages. Initially, the characterization of MH-S binding properties was performed. However, the principle objective with the MH-S cell line was to isolate CR3-negative populations of MH-S as a means to investigate the role of CR3 in the association of MTB. MTB association assays showed that the association of MTB with MH-S cells was comparable with mouse AM. In the presence of serum, increased MTB binding by MH-S cells was also mediated by a heatlabile serum component and competitive inhibition assays using anti-CR3 monoclonal antibodies showed that CR3 on MH-S cells is a quantitatively important receptor for MTB. However, attempts to isolate stable CR3-negative populations of MH-S cells using immunoselection were not successful and therefore, I was unable to use the MH-S cell line as a model to further characterize the role of CR3 in MTB pathogenesis.