INVESTIGATING EFFECTS OF COCR NANO PARTICLES ON THE IMMUNE SYSTEM

Abstract

Background: Increased use of metal on metal (MOM) hip replacements has stimulated interest in immunological effects of chronic CoCr elevation. Unlike metal-on-polyethelylene, MOM implants are associated with a perivascular infiltrate of lymphocytes & plasma cells. This may be the mode of failure of MOM implants. A reduction in CD8+ T lymphocyte counts associated with MOM implants has previously been described. CoCr therefore seems to affect the adaptive immune response even though it is not a proteinous antigen.

We therefore analyzed the effects of CoCr particles on T cells & B cells. We also analyzed it effects on dendritic cells, which are the key antigen presenting cells to T helper cells.

Methods: CoCr nano particles were produced by repetitive short spark discharges between electrodes of prosthetic CoCr alloy. Electron micrography & BET both confirmed nanoparticle size.

Dendritic cells (DCs) were harvested from mouse bone marrow & cultured in medium supplemented with GM-CSF for 6 days, generating DCs typically 80–90% CD11c+. These were incubated with CoCr in concentrations of 25, 10 & 2.5 μg/ml, for 24 hours, or lipopolysaccharide 1 μg/ml as a positive control. Following incubation, activation status of CD11c+ DCs was characterized by MHC Class II, CD40, CD80 & CD86 expression by FACS analysis.

T-Lymphocytes were harvested from mouse lymph nodes & cultured in medium without phenol red. These were incubated at 5 ×105 cells/well with either CoCr, conA (positive control) or CoCr + conA & repeated using 2.5 ×105 cells/well. Other positive controls (CD3 & CD 28) were studied in repeating the experiment. At 48 hours Almar Blue was added & further incubation for 24 hrs. Light absorbance at 570nm & 600nm was then used to determine T cell proliferation

B-Lymphocytes were harvested from the lymph nodes of mice which were only able to mount a B-cell reaction to Hen egg Lysozyme (HEL). These were incubated with medium with CoCr, HEL (positive control) or CoCr+ HEL. The concentration of the CoCr was varied between 25, 10 & 2.5 μg/ml. FACS analysis for markers of B cell regulation was performed after 48 hours incubation..

Results: CoCr did not significantly increase CD 40 expression on DCs, although such expression was increased significantly by lipopolysaccaride CoCr did not significantly up or down regulate B cells as compared to the effects of HEL. CoCr did inhibit proliferation of T-cells & this was more pronounced where the ratio of CoCr/cell density was higher.

Conclusion: Both dendritic & B cells are unaffected by CoCr in vitro. However, CoCr inhibited T cell proliferation. This demonstrates the observed reduction in CD + T cells are probably due to a direct effect of CoCr, & not mediated through another cell type. The perivascular response to MOM implants on the other hand probably requires cell interaction in an in vivo environment.