Macrophage mediated processing of an exogenous antigenic fluorescent probe: Time‐dependent elucidation of the processing pathway

Abstract

To elucidate time-dependent pathways and mechanisms involved in antigen processing, a fluorescent probe suitable to monitor several steps within this pathway was developed. Previous studies utilizing two-photon fluorescence microscopy with time resolved and intensity imaging demonstrated that the probe, fluorescein derivatized BSA, was localized to the endocytic system and degraded over an extended period of time. However, an additional method, flow cytometry, was required to monitor the kinetics of these intracellular events and to better assess the total cell population. Flow cytometric studies indicated that the antigen entered an acidic intracellular environment consistent with the endocytic system of the macrophage. Additional experiments suggested that minimal proteolytic degradation began 10 min after addition of the antigenic probe while extensive enzymatic degradation did not occur until 180-200 min. Inhibitor studies indicated that degradation of the probe was dependent upon both acidic pH and ATP synthesis as well as all four classes of proteases. Experiments involving specific protease inhibitors also revealed that various classes of proteases were active at different time points throughout the processing of the probe. By combining these results with additional kinetic data, a model for the sequence of events involved in the processing of FITC10BSA was proposed. More importantly, these studies represented some of the first time-dependent kinetic measurements of antigen processing in living cells.

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

“…D-isomers of poly-lysine were chosen for the following reasons: 1) it was generally known that eukaryotic cellular receptors did not recognize D-amino acids (19,26), and 2) utilizing D-polymers facilitated various fluorescence intensity, quenching, lifetime and kinetic measurements since the linear D-polymers were resistant to intracellular thiol reduction, acidic pH-mediated unfolding and proteasemediated proteolysis. These intracellular events played a major role in the processing of FITC-BSA and were shown to directly affect fluorescence measurements within the endocytic pathway (33,34,35). Selection of poly-D-lysine as a carrier eliminated these variables in fluorescence quantification and facilitated a focused investigation of the involvement of an aromatic hapten in macrophage receptor recognition, binding and internalization.…”

“…Murine macrophage cell line J774, provided by K.W. Kelley (University of Illinois, Urbana), was grown as previously described (34). Murine (BALB/c) myeloma B cell line SP2/0, provided by S. Miklasz (Immunological Resource Center, University of Illinois, Urbana), was grown in DMEM supplemented with 10% fetal calf serum and ampicillin-gentamycin antibiotics.…”

“…Experimental procedures for measuring binding and uptake of FITC-antigens and the set-up for flow cytometric measurements were previously reported (3,34). Saturation binding was determined in the presence of 3.0 mM amiloride and in the absence of serum.…”

“…However, at high FITC epitope densities (e.g. FITC 34 PDL 2 ) aromatic stacking (described below) between FITC residues probably contributed to energy transfer resulting in even shorter lifetimes of ϳ2.0 ns at pH 5.0. Fluorescence intensities and lifetimes of FITC-BSA and FITC-PLL increased upon in vitro enzymatic proteolysis or prolonged incubations with macrophage due to spatial separation of the FITC moieties on generated peptides resulting in alleviation of energy transfer and autoquenching (8,33).…”

“…Results obtained also validated the binding of the fluorescein hapten to a putative cellsurface receptor that possessed specificity for phenyl-like aromatic structures. FITC (isomer I) was reacted with the poly-D-lysine (PDL) polymers under basic conditions as previously described for BSA (34). Various degrees of poly-D-lysine derivatization were obtained by altering FITC concentration.…”