A Potential Role for Shed Soluble Major Histocompatibility Class I Molecules as Modulators of Neurite Outgrowth
Figure 4
Recombinant, soluble MHCI [sMHCI(1-298)] inhibits retina neurite outgrowth.
A) COS cells were transfected with a non-recombinant plasmid (lane 1) or one encoding Db heavy chain amino acids 1–298 which lacks a membrane anchor (lanes 2 and 3), and grown for 48 hrs in 35S methionine containing media. The media was harvested, a conformation-dependent anti-MHCI-Db (lanes 1 and 3) or anti-MHCI-Kk (lane 2) mAb was added, the bound proteins were collected on protein A sepharose beads and analyzed using SDS-PAGE and autoradiography. Image shows labeled immunoprecipitated proteins with the expected molecular weights of the Db heavy chain and β2m in lane 3. B) Retina neurite outgrowth in the presence of a dose range of sMHCI(1–298). N = 8 wells from 2 experiments. There was a significant main effect of dilution concentration on average neurite outgrowth (F(4,34) = 9.54; p<0.001). This was further confirmed by the systematic increase of neurite outgrowth with subsequent reductions in dilution concentration (R2 = 0.3215; F(1,37) = 17.53; p<0.001). Representative composite images of an retina explant cultured with (C) conditioned media from control COS cell cultures, or (D) conditioned media from sMHCI(1–298) expressing COS cells (image shown is from a culture with 500 pM sMHCI(1–298)). Composite photos are shown at the same magnification. E) Average neurite outgrowth from retina explants grown in media alone (open bar), media to which conditioned media from COS cells transfected with a control plasmid (hatched bar), or a plasmid encoding Db(1–298) (black bars) was added. The indicated mAb was added to some cultures. N = 23–27 wells from 4 experiments. **p<0.01, ***p<0.002 by Student's t-test.