INBRE

Chemotactic cytokines (chemokines), such as CC chemokine ligand 3 (CCL3) (Macrophage inflammatory protein 1 alpha) (MIP1-α), are essential for leukocyte extravasation at inflammatory sites. T cells express the greatest number of distinct chemokine receptors, which mediate the recirculaton of specific T cell subpopulations. A CD4+ T helper type 1 (Th1) response is required to resolve chlamydial genital tract (GT) infections and predominates in the inflamed GT, but also may mediate pathological sequelae. In this proposal, we will evaluate the role of CCL3 in a murine model of chlamydial genital infection using CCL3 knockout (KO) mice (CCL3-/-). Our preliminary data indicates that the duration of genital infections is significantly reduced in CCL3 deficient mice and that these mice have higher levels of the Th1 cytokine interferon gamma (IFN-γ), in genital secretions and draining lymph node supernatants, when compared to wild-type controls. Moreover, mice lacking CCL3(-/-) have higher levels of the IgG2a subclass, which is associated with a Th1 response, in GT secretions. We propose to find out why mice lacking CCL3 have a less intense primary genital infection and are mounting a stronger Th1 response relative to syngeneic controls. To this end, we propose to evaluate the phenotype of leukocytes in the GT by flow cytometry. It has been reported that CCL3 preferentially recruits CD8+ cells to inflammatory sites. If this hypothesis is true, we should see an increased CD4+/CD8+ ratio in the GT of CCL3(-/-) compared to wildtype controls and this may, in part, explain the shorter course of infection in our CCL3 KO mice. A recently developed anti-CCR3 antibody will be used to stain leukocyte subsets in the GT and we may see a higher level of expression of CCR3 on CD8+ cells relative to CD4+ cells. In order to determine if homing receptor expression on leukocyte subsets is altered, to compensate for the loss of CCL3, we will stain mononuclear cells isolated from the GT with antibodies directed against various integrin and selectin molecules, as well CD44. Additionally, we will collect genital tract secretions and draining lymph node supernatants and determine chemokine concentrations by enzyme linked immunosorbent assays (ELISAs), particularly regulated-on-activation, normal T-cell-expressed-and-secreted (RANTES), which shares a receptor with CCL3. To determine the frequency of Th1 and T helper type 2 (Th2) cells in the GT and draining lymph nodes we will employ the enzyme-linked immunospot (ELISPOT) assay. Understanding the mechanisms by which protective T cell subpopulations are recruited to the GT, following chlamydial infection, would be useful in developing efficacious vaccination protocols. Future proposals involve evaluating secondary infection and pathology in CCL3 KO mice.

Back

Updated 10/31/2005

The Arkansas INBRE is Supported by a grant from the National Institutes of Health
and the National Center for Research Resources (P20 RR-16460).

Please contact Caroline Miller Robinson regarding questions or comments about this site or our program. For more information about the University of Arkansas for Medical Sciences visit http://www.uams.edu.