No abstract

The hyposideremia of inflammation was found to be based on a three-step mechanism involving lactoferrin, the iron-binding protein from the specific granules of neutrophilic leukocytes. (a) Lactoferrin is Released from Neutrophils in an Iron-Free Form. When phagocytosis was induced in neutrophils by zymosan or bacteria, lactoferrin was recovered in the incubation medium together with other constituents of the specific granules, such as alkaline phosphatase and lysozyme. Lactoferrin extracted from leukocytes was able to bind the amount of iron corresponding to its theoretical iron-binding capacity. After injection of endotoxin into rats, lactoferrin was detected in various tissues where it was normally absent, or in the plasma when the reticuloendothelial system (RES) had previously been blocked by injections of India ink or aggregated albumin. (b) Lactoferrin is Able to Remove the Iron from Transferrin. Significant exchange of iron from transferrin to lactoferrin was observed in vitro only at a pH below 7.0 or in the presence of a high concentration of citrate. However, the fast elimination of lactoferrin in vivo, when saturated with iron, might account for the observed transfer of iron to endogenous or administered apolactoferrin. Intravenous injection of human apolactoferrin into rats caused a marked decrease of the plasma iron level. The kinetics of this process, as well as controls with other proteins, ruled out the possibility of a secondary inflammatory effect due to phlogogenic contaminants. (c) Fe-Lactoferrin is Taken-up by the RES. By immunofluorescence, lactoferrin was shown to be bound and ingested by monocytes. The rate of elimination of human Fe-lactoferrin injected into rats was particularly fast when compared to that of human apolactoferrin, succinylated Fe-lactoferrin, or other human proteins. Blockade of the RES slowed down the rate of clearance of Fe-lactoferrin and was also found to retard the elimination of endogenous rat lactoferrin released by endotoxin. These experiments suggest the existence of specific receptors for Fe-lactoferrin on the membrane of macrophages.

Human iron-saturated Lf (FeLf), which was labeled with 125I or 50Fe, was found to combine with the membrane of mouse peritoneal cells (MPC) which consisted of 70% macrophages. The following experimental data suggested the involvement of a specific receptor. (a) The binding of FeLf to MPC reached a saturation point. (b) The binding of radioactive FeLf was inhibited by preincubating the cells with cold FeLf but not with human Tf, human aggregated and nonaggregated IgG, or beef heart cytochrome c (c) Succinylation and carbamylation of FeLf resulted in a loss of its inhibiting activity on the binding of radioactive FeLf. Removal of neuraminic acid from FeLf increased its inhibitory activity. (d) The ability of apoLf to inhibit the binding of FeLf to MPC was significantly lower than that of FeLf. The existence of a Lf receptor capable of concentrating Lf released from neutrophils on the membrane of macrophages could explain the apparent blockade of the release of iron from the reticuloendothelial system, which accounts for the hyposideremia of inflammation. A receptor for FeLf was also found on mouse peritoneal lymphocytes. The affinity constant of FeLf for both lymphocytes and macrophages was 0.9 X 12(6) liter/mol. Howerver, macrophages bound three times more FeLf molecules (20 X 10(6)) per cell than did lymphocytes (7 X 10(6)).

Enhancement by IgM rheumatoid factor of in vitro ingestion by macrophages and in vivo clearance of aggregated IgG or antigen-antibody complexes*Polyclonal as well as monoclonal IgM rheumatoid factors (RF) markedly enhanced both the attachment and ingestion of heat-aggregated human IgG (HAG) by mouse alveolar macrophages, mouse peritoneal macrophages and human peripheral monocytes. This effect depended upon the integrity of the Fc fragment of the HAG as shown by the experiments performed with reduced and alkylated HAG. Marked differences in the stimulation index were observed between the five tested RF.When we compared the effect of complement (C) and/or RF on the total amounts of HAG cleared from the medium, i.e. the sum of the HAG associated with the cells and the digestion products released in the medium, the addition of C alone resulted in a 10-fold increase, the addition of RF alone in a 20-fold increase, and the addition of both RF and C in a 10-fold increase. Apparently, the effect of RF was mainly on the binding of HAG to macrophages, whereas C mainly induced ingestion as indicated by the increased release of degradation products.Antigen-antibody (AgAb) complexes were prepared with 1251-labeled human transferrin as antigen and mouse anti-transferrin serum at various Ag/Ab ratios. Without RF, a significant uptake occurred up to a 3-fold Ag excess. RF caused a 3-fold increase of the uptake of complexes prepared at equivalence, but an 80-fold increase of the uptake of the complexes prepared in 1 0-fold Ag excess.Gradient ultracentrifugation of the AgAb complexes showed that the enhancement of the uptake due to RF was related to the increase of the sedimentation rate of the complexes. However, an excess of RF decreased its enhancing effect on endocytosis.One hour after the intraperitoneal injection of complexes in 10-fold Ag excess, the radioactivity of the peritoneal cells was 14 times higher when the complexes were pre-incubated with RF than with complexes not preincubated with RF. Similar complexes were also injected intravenously. The 13 S fraction which was visible on the ultracentrifugation profile in the absence of R F was eliminated very slowly, whereas the "heavy" fraction (> 30 S) which appeared after addition of RF, was cleared from the plasma in 2 5 sec.