Copper and innate immune system

This post explores how how a cupric nicotinic acid chelate might enhance macrophage phagocytosis of Gram negative bacteria.  The process is really not so simple after all. At each step we may ask the question, “Is copper a cofactor in proteins involved in the macrophage response to LPS?”

Copper binds to LPS

structural components of LPS and copper binding sites
Left, the overall structure of lipopolysaccharide. other components are shown to the right. Red stars mark possible places of Cu(I) binding.

We have extremely strong indication that copper binds LPS because Brubacher and others in the NC Bols laboratory found copper  contamination of some commercial preparations of LPS.  Results were published in 2003.  Their goal was to study reactive oxygen species generation from a fish macrophage cell line in response to LPS.

  • Baruch and coworkers observed that fish can withstand higher concentrations of LPS than mammals.
  • CD14 transfers LPS from the serum LPS binding protein to the cell membrane bound TLR4 receptor.
  • Trout have LBP1 and LBP2.
  • Trout don’t have  CD14  found in mammals.
  • They hypothesized lack of  CD14 would mean that fish macrophages would respond less to large doses of LPS

.  Along the way they discovered that one of their lots of LPS was contaminated with copper.

Copper contaminates some lots of LPS and may generate reactive oxygen species.
Two of three lots of LPS in the Brubacher study were contaminated with copper. Copper may have superoxide dismutase activity (top) or generate hydroxyl radicals.

Brubacher and co authors discussed the possibility that LPS naturally binds copper and other metals in the environment as a means of sequestering them.  These authors used a fluoescent probe to measure generation of reactive oxygen species in a trout and murine macrophage cell line and in a trout and human epithelial cell line.

Adapted from Table 2, (Brucbacher 2003).  The authors used a trout macrophage cell line.    Reactive oxygen species generation with an equivalent amount of copper was not performed.  The studies that were performed were in serum free medium so that LBP and CD14 were not involved.

“Endotoxin clearly did not cause increased ROS production in RTS11 cells, however, as copper-free LPS preparations had little effect on oxidation of H2DCF. Furthermore, removal of Cu-contaminated LPS resulted in a reversion to basal levels of H2DCF oxidation.” (Brubacher 2003).  The authors did not present data of equivalent amounts of copper without the LPS to confirm no participation of TLR4 pathways.  The human epithelial cell line, HT-29, has been reported to express TLR4 receptors in recent literature.  A possibility remains that copper bound LPS can bind to TLR4 unassisted.

The issue of LPS inducible nitric oxide synthase

The authors used inhibitors of

  1.  Superoxide generation from complex 1, which can be converted to hydrogen peroxide…. that can be converted to highly reactive hydroxyl radicals if copper bound to LPS gets it first.
  2. LPS inducible nitric oxide synthase which can produce nitric oxide… which can react with superoxide to produce peroxynitrite…



Both hydroxyl radicals and peroxynitrite can react with H2DCF to produce fluorescent DCF.  If it were not for the iNOS, we could conclude that everything could be explained by contaminating copper.  Perhaps contaminating copper might make it easier for LPS to bind to its receptor TLR4 without the usual helper protein(s).


A review of the literature of LPS and pro-inflammatory signalling…. and a look at Cu(I)…


LPS binding protein (LBP) is a 53 kDa  glycosylated protein that binds to the Lipid A portion of LPS.  It facilitates the transfer of LPS to CD14.  In addition to its four glycosylaton site, it also has a sisulfide bond.

CD14 is a solenoid shaped glycoprotein with a hydrophobic pocket for binding to LPS, which it  delivers to TLR4.


TLR4    the ancestral (fish) vertebrate LPS-response machinery is CD14- independent (Brubacher 2003).  Even with copper bound to the LPS, would fish macrophages generate.  There is no CD14 in trout




Brubacher JL, DeWitte-Orr SJ, Zorzitto JR, Playle RC, Bols NC.(2003) Redox-active metals in commercial preparations of lipopolysaccharide: implications for studies of cellular responses to bacterial products. Cell Microbiol. ;5(4):233-43.  Free Paper

Płóciennikowska A, Hromada-Judycka A, Borzęcka K, Kwiatkowska K. (2015) Co-operation of TLR4 and raft proteins in LPS-induced pro-inflammatory signaling. Cell Mol Life Sci. 72(3):557-581.  Free Paper



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