The suppressive effect of ferritin on macrophage colony-stimulating factor-dependent monocytopoiesis is cytokine mediated

An Ala-Lys-Pro-Arg [(Ala¹)-tuftsin) containing 14-Mer peptide (Glu-Thr-Val-Ile- Met-Lys-Ala-Lys-Pro-Arg-Ala-Asn-Phe-Pro) was synthesized representing a reported carboxyl terminus of acidic isoferritin. This peptide, like (Ala¹)-tuftsin and rat liver ferritin (RLF), inhibited macrophage colony-stimulating factor (M-CSF) stimulated colony formation by murine marrow-derived progenitors responding to the growth factor and bacterial lipopolysaccharide (LPS). Peptide-specific immunoglobulin G (IgG) from a rabbit immunized with the 14-Mer peptide neutralized ferritin inhibition of in vitro 2-signal-dependent progenitor clonal proliferation. Anti-ferritin IgG similarly neutralized the inhibitory effect of RLF but did not neutralize peptide inhibition of in vitro 2-signal-dependent progenitor clonal proliferation. Competitive and non- competitive enzyme-linked immunoassays (ELISAs), as well as Western immunoblot, demonstrated the anti-14-Mer IgG and anti-ferritin IgG only reacted with their respective antigen. Depletion of adherent cells before marrow cell culture significantly reduced the inhibitory activity of ferritin but did not reduce 14-Mer peptide inhibition of in vitro 2-signal-dependent progenitor clonal proliferation. Only adherent bone marrow cells or P388D₁, cells treated with a combination of M-CSF and ferritin produced inhibitory supernatant medium that was neutralized by anti-14-Mer IgG, but not anti- ferritin IgG. Studies involving recombinant human acidic isoferritins demonstrated intrinsic ferroxidase activity was necessary for production of anti-14-Mer IgG neutralizable inhibitory activity. High resolution molecular sieve chromatography of the inhibitory adherent marrow cell supernatants resolved two peaks of 51 kDa and 31 kDa. The inhibitory activity in both peaks was neutralized by anti-14-Mer IgG, but not anti-ferritin IgG. High resolution molecular sieve chromatography of ammonium sulfate precipitated and affinity purified P388D₁ cell derived inhibitory activity resolved two peaks of 58 kDa and 30 kDa. As with inhibitory activity derived from adherent bone marrow cells, the P388D₁, cell derived inhibitory peaks were neutralized by anti- 14-Mer IgG, not anti-ferritin IgG. The ferritin/M-CSF induced inhibitory activity was not further characterized, although identity with interleukins-6 and -8, tumor necrosis factor-a, leukemia inhibitory factor, transforming growth factor-β, and interferon-α/β could be eliminated. The results indicate ferritin inhibition of in vitro 2-signal- dependent clonal proliferation is medated by an endogenously produced inhibitor(s), that shares antigenic similarity with the (Ala¹)-tuftsin-containing 14-Mer peptide and that adherent marrow cells, most likely monocytes or macrophages, produce the endogenous inhibitor(s) in response to M-CSF and ferritin in combination. As evidenced by its in vitro biological activity, the inhibitor(s) may be beneficial in the treatment of cancer and may play an important role in regulating inflammatory processes.