Characterization of two kinds of lactotransferrin (lactoferrin) receptors on different target cells.

Lactotransferrin (Lf), an iron-binding glycoprotein present as a major component in the specific granules of human neutrophilic granulocytes is released in the blood during the acute phase of infection and participates in the regulation of the host-defence mechanisms. Our previous observations (Mazurier et al., 1989) showing i) that the activation by PHA of T-lymphocytes induces the appearance at the cell surface of Lf-receptors which are absent from the membrane of resting lymphocytes and ii) that Lf becomes a growth factor for the activated lymphocytes, led us to undertake a series of researches on the presence of Lf receptors at the surface of different blood cells. Characterization of Lf receptors was performed by flow cytofluorimetry using either Lf labelled on its glycan moiety with fluorescein or purified anti-lymphocyte Lf receptor antibodies. High affinity receptors for Lf were characterized only at the surface of human activated lymphocytes and of non-activated platelets. These two receptors possess common physicochemical properties and antigenic epitopes. Low affinity receptors for Lf were characterized on monocytes, eosinophils and neutrophils. These receptors are immunologically different from those found on activated lymphocytes and on non-activated platelets. Cell-lines of human lymphocyte T and megakaryocyte possess lactotransferrin receptors whose properties are similar to those found on peripheral blood cells. The soluble form of the receptor identified in the lymphocytes T culture medium possesses a molecular mass close to that of the membrane receptor suggesting that the cytoplasmic tail of the receptor should be very short.

Effect of intracellular iron depletion by picolinic acid on expression of the lactoferrin receptor in the human colon carcinoma cell subclone HT29-18-C1.

A lactoferrin receptor has been found on the brush-border membrane of intestinal epithelial cells of several species, including humans. A role for this receptor in intestinal iron absorption, which is well regulated in response to body iron stores, has been proposed. We have investigated the effect of intracellular iron depletion by picolinic acid, an iron chelator, on the cell surface binding of human lactoferrin to human enterocytes and its intracellular uptake, using HT29-18-C1 cells, an enterocyte-like differentiable cell line. The confluent cells exhibited 5.8 x 10(6) specific binding sites per cell for diferric human 125I-labelled lactoferrin with relatively low affinity (Kd 8.4 x 10(-7) M). The addition of picolinic acid to the culture medium resulted in a concentration- and time-dependent increase in lactoferrin binding that was correlated with a decrease in intracellular iron content. The maximum effect of picolinic acid on lactoferrin binding (approx. 2-fold increase), which appeared between 12 and 18 h after its addition, was obtained at a picolinic acid concentration of 2 mM. Scatchard analysis showed that the enhanced lactoferrin binding resulted from an increase in the number of lactoferrin receptors rather than an alteration in the binding affinity for lactoferrin. The time-dependent effect of picolinic acid was completely abolished in the presence of 1 microM anisomycin, a protein synthesis inhibitor, indicating that ongoing protein synthesis is involved in this effect. The enhanced lactoferrin binding induced by picolinic acid produced an increase of approx. 30% in the uptake of lactoferrin-bound 59Fe, indicating the existence of functional receptors. These results suggest that biosynthesis of lactoferrin receptors in intestinal epithelial cells can be regulated in response to the levels of intracellular chelatable iron, consistent with intestinal iron absorption dependent on body iron stores.

Apical-to-basolateral transepithelial transport of human lactoferrin in the intestinal cell line HT-29cl.19A.

The role of human lactoferrin (hLf) in alimentary iron absorption across intestinal cells was explored using a human differentiated colon carcinoma cell line, HT-29cl.19A. The apical surface of HT-29cl.19A cell monolayers exhibited 1.5 x 10(6) specific binding sites for hLf per cell, with a dissociation constant of 8.3 x 10(-7) M. The apical-to-basolateral transport of 125I-labeled hLf (125I-hLf) or 125I-59Fe-labeled hLf (125I-[59Fe]hLf) was investigated using filter-grown HT-29cl.19A cell monolayers mounted in Ussing chambers. Transport of total (intact plus degraded) hLf, measured by the 125I flux, was not saturable up to an apical hLf concentration of 12.5 microM, whereas immunoreactive hLf transport measured by enzyme-linked immunoabsorbent assay was saturable at 3.75 microM. Lowering the temperature to 4 degrees C reduced the total and immunoreactive hLf fluxes by 77% and 90%, and colchicine (500 microM) reduced these fluxes by 30% and > 97%, respectively. These results indicate that both types of transport are transcellular. Electrophoretic analysis revealed that 125I-hLf transported to the basal compartment consisted of largely degraded fragments (< 2 and 10-20 kDa) and a small portion of intact hLf. At an apical concentration of 3.75 microM diferric 125I-[59Fe]hLf, the immunoreactive hLf flux (64.6 +/- 14.3 ng.h-1.cm-2) constituted approximately 12% of the total hLf flux (552.0 +/- 61.6 ng.h-1.cm-2) and was similar to the [59Fe]hLf-equivalent flux (77.0 +/- 12.3 ng.h-1.cm-2).(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-lactoglobulin suppresses melanogenesis in cultured human melanocytes.

The effects of whey proteins from bovine milk on melanogenesis in cultured human melanocytes were examined. Among the major protein components of milk whey including beta-lactoglobulin (BLG), alpha-lactalbumin, serum albumin, and IgG, only BLG exhibited the depigmenting effect at a concentration of 1 mg/ml. Also, BLG suppressed the activity of tyrosinase in these cells. Retinol, to which BLG is known to bind, slightly increased the pigmentation of the cells at concentrations in the range of 1-100 nM, and retinoic acid, a metabolite of retinol, exhibited a strong pigmentation-promoting effect within the same concentration range. Treatment of the cells with 1 mg/ml BLG completely abrogated the pigmentation induced by these A vitamins. These results demonstrate a novel biological activity of BLG and suggest that this activity is dependent on its ability to bind retinol.

A high-Mr glycoprotein fraction from cow's milk potent in inhibiting replication of human rotavirus in vitro.

Rotavirus is the major cause of infectious diarrhea in infants and young children all over the world. We have found that a high-M(r) glycoprotein fraction from cow's milk is potent in inhibiting replication of human rotaviruses in vitro. Since the activity seems to be unique and specific, this fraction may be useful as a novel agent for treatment or prevention of rotavirus diarrhea.