FITC-poly-D-lysine conjugates as fluorescent probes to quantify hapten-specific macrophage receptor binding and uptake kinetics

Abstract

A series of fluorescein derivatized poly‐D‐lysine (FITC‐PDL) probes were used to elucidate the role of fluorescein in receptor binding of fluorescein‐conjugated macromolecules to J774 murine macrophages. Poly‐D‐lysine served to eliminate receptor recognition of the carrier due to the biologically inert nature of the D‐isomer. This concept enabled the focused investigation of the role played by fluorescein in receptor recognition, binding and internalization. Results revealed dependency of cellular uptake on polymer concentration, hapten density and accessibility. The results differed from those previously obtained with FITC‐BSA in that saturating fluorescein densities on the poly‐D‐lysine polymer resulted in diminished rates of uptake by macrophages. Receptor‐mediated endocytosis via clathrin‐coated pits was concluded based on results that showed inhibition of FITC‐PDL uptake by intracellular K+ depletion but not by the macropinocytosis inhibitor, amiloride. Further, FITC‐PDL was found to inhibit the endocytic uptake of FITC‐BSA suggesting competition between the two probes at the level of a macrophage receptor. Association rates (kon) for binding to the macorphage surface were measured for the various FITC‐PDL probes based on fractional receptor occupancies. Results are discussed on the basis of receptor recognition of fluorescein in J774 macrophages and the requirements for this recognition which include appropriate spacing and accessibility of the hapten moieties to facilitate receptor crosslinking. Cytometry 31:110–124, 1998. © 1998 Wiley‐Liss, Inc.

Dinuclear Alkylamine Platinum(II) Complexes of [1,2‐Bis(4‐fluorophenyl)ethylenediamine]platinum(II): Influence of Endocytosis and Copper and Organic Cation Transport Systems on Cellular Uptake

“…It is well known17, 18 that macropinocytotic processes, a distinct form of endocytosis, depend on a sodium gradient and can be blocked by amiloride and N ‐ethyl‐ N ‐isopropylamiloride (EIPA) as inhibitors of a Na + –H + exchanger 14. 18 Furthermore, as macropinocytosis is dependent on F‐actin microfilament rearrangement, the inhibition of F‐actin elongation by cytochalasin D, and the wortmannin‐mediated inhibition of phosphoinositide 3‐kinase (a key enzyme in the downstream signaling of macropinocytosis) blocked macropinocytosis efficiently 15. 17, 19, 20…”

Tracing heme in a living cell: hemoglobin degradation and heme traffic in digest cells of the cattle tickBoophilus microplus

“…Whatever the molecular nature of the digest cell receptor for hemoglobin, the existence of a distinct apparatus -receptor, cell compartments and possibly proteases and membrane transporters -dedicated to digestion of hemoglobin can be understood as a protection against the potential toxicity of the heme group, and it illustrates the complexity of adaptation to digestion of hemoglobin. In the case of mammalian macrophages, several other protein ligands are taken up by endocytosis (Cherukuri et al, 1998), but the possibility of a distinct path for hemoglobin hydrolysis has not been investigated.…”

Kinetics and intracellular pathways required for major histocompatibility complex II–peptide loading and surface expression of a fluorescent hapten‐protein conjugate in murine macrophage

“…By taking advantage of each of these properties, a kinetic model for the events required for MHC II–peptide loading and surface expression in murine macrophage was possible. Briefly, the antigenic protein was internalized via receptor‐mediated endocytosis or pinocytosis 50 –52 . Upon internalization, the probe entered a mildly acidic environment within the cell, and by 5 min, proteolytic cleavage of the probe began within early endosomal organelles ( Figs 1b and 3).…”

“…Since antigen is internalized into APC via several mechanisms, it was not suprising that multiple intracellular pathways and organelles would be involved in this process 29 . For example, a novel macrophage cell surface receptor specific for auromatic phenyl‐ring containing compounds was recently purified and characterized biochemically 50 –53 . The receptor significantly enhanced both the intracellular uptake and concentration of the antigen 36 .…”