Resolution of rabbit polyclonal anti-fluorescein Fab (IgG) fragments into subpopulations differing in affinity and spectral properties of bound ligand.

Fab fragments derived from ten different IgG populations of hyperimmune rabbit polyclonal anti-fluorescein antibodies were further resolved into subfractions based on differences in time-dependent dissociation from an FITC-adsorbent in the presence of 0.1 M fluorescein at 4 degrees C. Fab fragments separated into subpopulations based on specific dissociation times of 0.1 day, 1.0 day, 10 days and 100 days from the adsorbent. Finally, after the 100 days elution step incubation with 6.0 M guanidine-HCl was included to determine total protein concentration of specific anti-fluorescein Fab fragments. Yields of specifically eluted Fab fragments ranged from 12.7 to 84.1% of the total Fab population originally incubated with the adsorbent. All Fab polyclonal populations and subpopulations analyzed quenched the fluorescence of the bound ligand by 90% or greater. None of the plots of protein concentration versus percent yield of the total specific antibody obtained for each of the five resolved fractions constituting a specific polyclonal population conformed to Gaussian distributions. All resolved Fab subpopulations retained bound fluorescein ligand that exhibited significant bathochromic shifts in absorbancy. Based on the extent of the red-shift the antibodies segregated into one of two general spectral families showing either a peak shift to 505-507 nm or to 518-520 nm. The red-shift to 518-520 nm appeared unique to rabbit anti-fluorescein antibodies, since corresponding large shifts have not been observed with antibodies derived from other species (e.g. mouse, rat, chicken, etc.). K(d) values determined for the resolved fractions confirmed a continuous progression in affinity from the 0.1day through the 100 days elution. Preliminary isoelectric focusing analyses revealed progressive selection for relatively more homogeneous fractions, especially in the 100 days resolved fraction.

A novel cellular interaction involving antigen-pulsed macrophage and antigen-specific B-lymphocytes.

Extensive documentation shows that macrophage efficiently present antigen to CD 4(+) T-cells in conjunction with the MHC II molecule. Previously, a novel fluorescent probe, FITC-BSA, was developed to analyze intracellular antigen processing and presentation pathways within viable peritoneal murine macrophage. The studies revealed fluorescein's accessibility to antibody binding when associated with peptides bound within the MHC II cleft. To determine if MHC II-fluoresceinated-peptide complexes on the surface of macrophage were also sufficient to stimulate antigen-specific B-cells, nylon wool-purified splenic B-cells from FITC-KLH injected BALB/c mice (H-2(d)) were co-cultured with antigen-pulsed macrophage. B-cell stimulation and antibody production was observed in the presence of FITC-BSA-pulsed macrophage, whereas, macrophage incubated in the presence of unlabeled BSA were not stimulated. Compared with control cells, similar levels of stimulation were detected following depletion of Thy 1.2(+) cells from nylon wool-based spleen cell preparations. Stimulation was inhibited upon preincubation with anti-fluorescein IgG antibodies. Stimulation was not measurable using B-cells derived from the naive mice. The interaction was inhibited upon addition of MHC II specific antibodies and leupeptin, a microbial product that inhibits MHC II-peptide complex formation. Importantly, antibody production was not observed in the presence of antigen-pulsed macrophage from H-2(b) mice. Moreover, B-cell stimulation via this pathway was dependent upon antigen concentration as well as the cell to cell ratio.

Virus inactivation by anilinonaphthalene sulfonate compounds and comparison with other ligands.

Bis-(8-anilinonaphthalene-1-sulfonate) (bis-ANS) causes inactivation of vesicular stomatitis virus (VSV) at micromolar concentrations while butyl-ANS and ANS are effective at concentrations one and two orders of magnitude higher, respectively. VSV fully inactivated by the combined effects of 10 microM bis-ANS and 2.5 kbar hydrostatic pressure elicited a high titer of neutralizing antibodies. Incubation of VSV with >/=2 M urea at atmospheric pressure caused very little virus inactivation, whereas at a pressure of 2.5 kbar, 1 M urea caused inactivation that exceeded by more than two orders of magnitude the sum of the inactivating effects produced by urea and pressure separately. Measurements of bis-ANS fluorescence showed that increasing the urea concentration reduces the pressure required to disrupt the structure. We conclude that anilinonaphthalene sulfonate compounds inactivate VSV by a mechanism similar to that produced by pressure. The most effective antiviral compound was bis-ANS which can be used for the preparation of safe viral vaccines or as an antiviral drug eventually.

DNA hydrolysis by monoclonal autoantibody BV 04-01.

Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg2+. Catalysis was associated with BV 04-01 IgG, Fab, and single-chain-antibody (SCA) proteins. Cleavage of both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of A7C7ATATAGCGCGT2, as well as a preference for cleaving within CG-rich regions of dsDNA. Data on specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA, and two SCA mutants were used to model the catalytically active antibody site using the previously resolved X-ray structure of BV 04-01. The resulting model suggested that the target phosphodiester bond is activated by induction of conformational strain. In addition, the antibody-DNA complex contained a Mg2+ coordination site composed of the L32Tyr and L27dHis side chains and a DNA 3'-phosphodiester group. Induction of strain along with the metal coordination could be part of the mechanism by which this antibody catalyzes DNA hydrolysis. Sequence data for BV 04-01 V(H) and V(L) genes suggested that the proposed catalytic-antibody active site was germline-encoded. This observation suggests that catalytic activity might represent an important-rarely examined-function for some antibody molecules.

Intracellular mechanisms responsible for exercise-induced suppression of macrophage antigen presentation.

In a previous study, we demonstrated that exhaustive exercise suppressed peritoneal macrophage antigen presentation (AP). In this study, we explored the intracellular mechanism(s) responsible for this suppression. Pathogen-free male BALB/c mice (8 +/- 2 wk) were randomly assigned to either home cage control (HCC) or exhaustive exercise stress (Exh, 18-30 m/min for 3 h/day) treatment groups. The mice underwent treatments for a period of 4 days during induced peritoneal thioglycollate inflammation. Elicited macrophages were harvested, purified, and incubated with chicken ovalbumin (C-Ova, 2. 5 and 10 mg/ml) for 18 h. After macrophages were washed, they were cocultured with C-Ova-specific T cells for 48 h at which time the supernates were harvested and analyzed via ELISA for interleukin (IL)-2 as an indication of macrophage AP. There was no significant (P > 0.05) difference in macrophage AP between cells fixed with paraformaldehyde vs. those that remained unfixed, suggesting that Exh did not affect production of soluble factors influencing macrophage AP (i.e., IL-1, IL-4, PGE(2)). The ability of macrophages to generate C-Ova immunogenic peptides was analyzed using FITC-labeled C-Ova, which shows fluorescence only when degraded intracellularly. There was a significant ( approximately 20%, P < 0. 05) suppression in fluorescence in the Exh compared with HCC, indicating a possible defect in the ability of macrophages from Exh to degrade C-Ova into immunogenic peptides. Macrophages were also incubated with C-Ova immunogenic peptide in a manner identical to that for native C-Ova. We found a similar suppression ( approximately 22-38%, P < 0.05) in macrophage AP using a C-Ova peptide when compared with native C-Ova in the Exh group, indicating reduced major histocompatibility complex (MHC) II loading and/or C-Ova-MHC II complex cell surface expression. In conclusion, these data indicate an intracellular defect in the macrophage antigen processing pathway induced by Exh.

Interaction of dual-specific autoantibodies with dsDNA and a synthetic dimer peptide simulating the hinge region of IgG2a molecules.

Anti-dsDNA autoantibodies and immune complex formation are major factors in SLE pathogenesis. Understanding stable immune complex formation is critical in deciphering mechanisms of autoimmune pathogenesis. Previous studies identified a subpopulation of murine lupus monoclonal autoantibodies that exhibited dual specificity (anti-DNA and anti-IgG2a hinge) and formed stable immune complexes [J. Mol. Rec. 10(1997)225]. Two monoclonal autoantibodies, BV 17-45 and BV 16-13, were extensively studied because of their dual specificity. To quantitatively assess the role of each specificity in the formation of stable immune complexes, studies were performed to determine binding affinities for various sized dsDNA fragments (21, 43, 84, and 114 bp) and the covalent dimer of a nine amino acid hinge peptide. Results characterizing BV 17-45 showed that the affinity for dsDNA directly correlated with increased dsDNA size. Results with BV 16-13 revealed a generally lower affinity for the various dsDNA fragments. Binding inhibition studies, using a covalently linked dimer of a nine amino acid synthetic hinge peptide as an inhibitor of the antibody-43 bp dsDNA interaction, yielded relative affinities for the anti-hinge activity. Binding affinities for the synthetic hinge specificity were lower than affinities measured for the anti-dsDNA activity. Collectively, the binding and inhibition studies provided insight into the correlation between dual specificity and avid immune complex formation. A model was proposed based on the concept that large dsDNA fragments caused localization of the dual-specific antibodies through the anti-dsDNA activity, thereby facilitating subsequent binding and cross-linkage via the anti-hinge specificity. These synergistic interactions resulted in the formation of avid immune complexes.

Biochemical purification and partial characterization of a murine macrophage surface receptor possessing specificity for small aromatic moieties including fluorescein.

Binding properties and requirements for internalization of hapten-protein antigens, such as fluorescein-polyderivatized bovine serum albumin (FITC-BSA) and poly-D-lysine (FITC-PDL), by murine macrophage was consistent with surface receptor recognition of fluorescyl moieties (Cherukuri et al., Mol. Immunol. 34, 21-32, 1997; Cherukuri et al., Cytometry 31, 110-124, 1998). Ligand binding properties of the putative macrophage receptor pointed toward specificity for various aromatic moieties including phenylalanine, phenyloxazolone and fluorescein (Cherukuri and Voss, Mol. Immunol. 35, 115-125, 1998). Purification of the hapten-recognizing receptor from J774 macrophage cells involved subcellular fractionation of plasma membrane fractions, and affinity chromatography of solubilized membranes employing a phenyl-Sepharose adsorbent with subsequent specific elution of receptor using fluorescein ligand. The final product was a protein with a molecular mass of approximately 180 kDa. Characterization of the purified receptor involved absorption and fluorescence spectroscopy, circular dichroism, fluorescence quenching analyses, various ligand binding assays and an immunological analysis. Spectroscopic analyses revealed that the receptor possessed aromatic amino acids while circular dichroism suggested significant alpha-helical secondary structure. Binding specificity of the purified receptor was confirmed in a spectrofluorometric assay where the fluorescence of fluorescein ligand was quenched approximately 97%. Finally, specific binding activity of the receptor with FITC-BSA was demonstrated in Western blot analysis under native conditions. Receptor purity was confirmed in amino acid sequencing analysis when the amino-terminal residue was found to be totally blocked. Results are discussed in terms of the possible identity of the isolated macrophage receptor and its biological-immunological role.

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

A fluorescent antigen, FITC10BSA, that is sensitive to several of the biochemical processes involved in antigen processing was constructed. In combination with both flow cytometry and subcellular fractionation, the unique probe provided new details regarding the kinetics and intracellular pathways involved in antigen processing in murine macrophage. These studies suggested that macrophage utilized multiple vesicles as opposed to a few specific organelles for major histocompatibility complex (MHC) type II-peptide loading and transport. Although newly formed MHC II-peptide complexes were detected in cathepsin D-positive, lysosomal associated membrane glycoprotein (LAMP-1)-positive lysosomes, MHC II-peptide loading also occurred in transferrin receptor-positive endosomes. Interestingly, MHC II-fluoresceinated complexes were only observed in transferrin receptor-positive organelles as opposed to MHC II-unlabelled peptide complexes which were detected in traditional early lysosomal compartments. More importantly, MHC II-peptide complexes were monitored in light transferrin receptor-positive fractions following their initial appearance in dense endosomal/lysosomal fractions. Control experiments suggested that these complexes represented intermediates in the process of migrating to the cell surface through a retrograde pathway within the macrophage.

A novel macrophage receptor enhances MHC II-peptide loading and surface expression of a hapten-protein conjugate.

A receptor possessing specificity for fluorescein was previously identified on murine macrophage. The goal of the present study was to determine if this receptor influenced MHC II-peptide loading and surface expression of a hapten-protein conjugate within murine macrophage. Although inhibition of fluid-phase pinocytosis had no detectable effect, lower levels of intracellular MHC II-peptide complexes were observed upon inhibition of receptor-mediated endocytosis. Moreover, lower levels of surface expressed MHC II-fluoresceinated peptide complexes were also detected. Following subcellular fractionation experiments, it was revealed that the receptor altered the endocytic trafficking of the antigen within the cell. Namely, degraded antigen and MHC II-peptide complexes were not observed in dense transferrin receptor positive, cathepsin D positive, LAMP-1 positive organelles upon inhibition of the receptor. Previous studies also suggested that this receptor enhanced MHC II-peptide loading by concentrating high levels of antigen to endocytic organelles. The implications of these findings on subsequent development of the immune response were also discussed.

Two dual-specific (anti-IgG and anti-dsDNA) monoclonal autoantibodies derived from the NZB/NZW F1 recognize an epitope in the hinge region.

The anti-IgG properties of two dual-specific (anti-dsDNA and anti-IgG) monoclonal NZB/NZW F1-derived autoantibodies, BV 17-45 and BV 16-13, were studied to resolve the location and possible commonality of the IgG epitope. To determine if BV 17-45 and BV 16-13 recognized the same IgG epitope, the relative temperature sensitivity of the conformational IgG epitopes were evaluated using the conformational sensitive immunoassay. Comparison of the temperature sensitivity of the conformational immunoglobulin epitopes over a temperature range of 25-100 degrees C suggested that the epitope recognized by BV 17-45 was the same as the IgG epitope recognized by BV 16-13. Further studies with papain- and pepsin-generated F(ab')2, Fab, and Fc fragments of BV 17-45 and BV 16-13 revealed that the dual-specific autoantibodies BV 17-45 and BV 16-13 both bound an epitope in the hinge region of the IgG molecule. The potential correlation between these studies and the pathogenic nature of dual-specific autoantibodies is discussed.

Analysis of rates of receptor-mediated endocytosis and exocytosis of a fluorescent hapten-protein conjugate in murine macrophage: implications for antigen processing.

A novel fluorescent hapten-protein conjugate was constructed to monitor the events required for CD 4+ lymphocyte recognition of antigenic proteins. Previous studies utilizing the probe demonstrated that the hapten-protein was localized to an acidic endocytic compartment within the macrophage and that the hapten-protein was sensitive to multiple intracellular events including enzymatic degradation, acidification, and disulfide bond reduction. More importantly, recent experiments indicated that efficient internalization of the probe was dependent upon specific recognition of the hapten. Therefore, the present report addressed the effect of receptor-mediated endocytosis upon the processing of the hapten-protein within murine peritoneal macrophage. These studies determined that the rate of endocytosis was significantly faster than the rate of exocytosis. Specifically, the rate of exocytosis was estimated to be 3.4 x 10(4)s-1 based on a unimolecular rate constant. Although at higher concentrations, a slightly slower rate was observed (1.9 x 10(4)s-1). This study also represented one of the first efforts to measure the intracellular concentration effect typically associated with receptor-mediated endocytosis. Experiments involving a radioactively labeled hapten-protein conjugate revealed that the probe was at 100-fold higher concentration within the endocytic vesicles when compared to the extracellular media. The intracellular mechanism involved in this phenomenon was discussed as well as the implications of these findings upon MHC II-peptide binding.

Effects of secondary forces on a high affinity monoclonal IgM anti-fluorescein antibody possessing cryoglobulin and other cross-reactive properties.

The effects of secondary forces on monoclonal IgM anti-fluorescein antibody 18-2-3 reactivity were investigated and the results correlated with similar studies characterizing anti-fluorescein mAbs 4-4-20 and 9-40. mAb 18-2-3 was considered an important model for further elucidation of secondary forces since it possessed ligand binding properties similar to mAb 4-4-20, such as a similar affinity, but due to a very different primary structure it was idiotypically and metatypically distinct. mAb 18-2-3 also possessed cryoglobulin (anti-Ig) and extensive cross-reactive properties (e.g. anti-phenyloxazolone) suggestive of an atypical anti-fluorescein active site. The reactivity of mAb 18-2-3 with model fluorescein-peptides was modulated by secondary forces in a manner that differed from both mAbs 4-4-20 and 9-40. Thus, the effects of secondary forces seemed to vary with each monoclonal antibody even though each of the immunoglobulins studied were specific for the same homologous ligand. Results indicated that secondary forces impacted immune complex stability, variable domain conformation and protein dynamics. Models were postulated to account for secondary effects on the mAb 18-2-3 active site relative to mAbs 4-4-20 and 9-40. Levels of hydration, active site architecture and local amino acid dynamics were among the models cited.

Ligand binding specificity of a macrophage surface receptor utilized by the fluorescein hapten for uptake into the endocytic pathway.

A series of compounds were tested as inhibitors of receptor-binding and uptake of fluorescein-derivatized antigens in primary peritoneal and J774 murine macrophage. Results were analysed with regard to the inhibitory potency of the tested ligands and revealed that the putative cell surface receptor preferentially recognized and bound aromatic structures containing phenyl rings. L-phenylalanine was found to be the most potent inhibitor among the ligands tested. Significant inhibition of FITC10BSA binding to macrophage was observed even at 10(-9) M concentration of specific monovalent ligands tested indicating that these ligands were bound by the putative macrophage receptor with high apparent affinity. Structural comparisons of the various inhibitors employed, demonstrated that accessible, unconjugated phenyl rings were bound by the putative receptor with high apparent affinity whereas non-phenyl derivatives were bound with either low apparent affinity or via non-specific interactions. Therefore, the fluorescein hapten appeared to utilize a receptor with specificity for an essential aromatic amino acid for gaining entry into the endocytic pathway of murine macrophage. Finally, the binding of the hapten was enhanced when polyvalent fluorescein-derivatized antigens were used as a result of receptor crosslinkage on the cell surface.

Spectral properties of fluorescein in solvent-water mixtures: applications as a probe of hydrogen bonding environments in biological systems.

Although fluorescein is a widely used fluorescent probe in the biosciences, the effect of solvent environment on its spectral properties is poorly understood. In this paper we explore the use of fluorescein as a probe of the state of hydrogen bonding in its local environment. This application is based on the observation, originally made by Martin (Chem. Phys. Lett. 35, 105-111, 1975), that the absorption maximum of fluorescein undergoes substantial shifts in organic solvents related to the hydrogen bonding power of the solvents. We have extended this work by studying the spectral properties of the dianion form of the probe in solvent-water mixtures. We show that the magnitude of the shift correlates with the alpha and beta parameters of Kamlet and Taft (J. Am. Chem. Soc. 98, 377-383; 2886-2894, 1976), which provide a scale of the hydrogen bond donor acidities and acceptor basicities, respectively, of the solvents. In solvent-water mixtures, these shifts reflect general effects of the solvents on the hydrogen bonding environment of the fluorescein through water-solvent hydrogen bonding and specific effects due to fluorescein-solvent hydrogen bonding. Indeed, both the absorption and fluorescence properties appear to be dominated by these effects indicating that the spectral shifts of the dianion can be used as an indicator of its hydrogen bonding environment. We discuss the application of fluorescein as a probe of hydrogen bonding in the microenvironment immediately surrounding the fluorophore, and we illustrate the effect with reference to the fluorescein-antifluorescein antibody complex where it appears that antibodies selected during the immune response possess binding sites that are increasingly dehydrated and hydrophobic.

Secondary force-mediated perturbations of antifluorescein monoclonal antibodies 4-4-20 and 9-40 as determined by circular dichroism.

Secondary forces have been defined as those interactions between antibody and antigen that occur external to the antibody active site. Previous investigations indicated that non-active-site secondary interactions can modulate immune complex stability and may influence antibody variable domain conformation and/or dynamics. To assess secondary force-induced perturbations of monoclonal antibodies 4-4-20 and 9-40 a series of monofluoresceinated peptides was reacted and the various interactions analyzed by circular dichroism (CD). The mAbs 4-4-20 and 9-40 vary by nearly 1000-fold in their respective affinities for the fluorescein ligand, yet both immunoglobulins are highly related at the primary structural (idiotype) level. Near-UV CD spectra were evaluated as well as the induced optical activity (visible CD) of the antibody-bound fluorescein moiety when covalently attached to various peptide carriers. Comparative spectral studies revealed significant differences in the near-UV CD spectra of mAbs 9-40 and 4-4-20 relative to the various peptide antigens and to one another. CD spectra were interpreted as reflecting differential secondary force-induced perturbations of the antibody variable domains as well as intrinsic differences between the two mAbs.

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

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 macrophage 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.

Effects of secondary forces on the ligand binding and conformational state of antifluorescein monoclonal antibody 9-40.

Biochemical interactions that occur external to the antibody active site have been termed secondary forces. Secondary forces are supplemental to interactions within the antibody active site (i.e., primary interactions) and can affect ligand binding efficiency as well as variable domain conformation. The antifluorescein antibody system has been determined to be a superior method for delineating primary from secondary interactive components due to the active site-filling properties of the fluorescyl ligand. To date, all studies of secondary forces within the context of the antifluorescein system have been with the high-affinity monoclonal antibody 4-4-20 (mAb 4-4-20) (Mummert & Voss, 1995, 1996, 1997). In order to determine the generality of experimental observations and proposed models, we investigated the effects of secondary forces on the antifluorescein mAb 9-40. In addition to assessing the results of former studies, mAb 9-40 possesses properties unique from those of mAb 4-4-20, namely, a decreased affinity for fluorescein and increased conformational dynamics relative to mAb 4-4-20 (Carrero & Voss, 1996). Results of fluorescein and intrinsic mAb 9-40 tryptophan quenching as well as differential scanning calorimetric (DSC) studies indicated that secondary forces modulated the conformational (metatypic) state in accordance with previous investigations with mAb 4-4-20. Unlike mAb 4-4-20, mAb 9-40 did not exhibit altered ligand binding efficiency due to the inclusion of secondary interactive components. Models were developed that proposed that the increased malleability of mAb 9-40 variable domains could account for functional differences in properties between mAb 9-40 and mAb 4-4-20.

Analysis of the intracellular processing of proteins: application of fluorescence polarization and a novel fluorescent probe.

Previous studies indicated that fluorescein derivatized bovine serum albumin was an ideal probe to monitor the time-dependent kinetics of antigen processing in the murine macrophage cell line J774. Whereas previous work focused on fluorescence intensity measurements, the present study relied on fluorescence polarization to dissect the local environment of the fluorescent hapten-protein within the endocytic system of the cell. A steady increase in both fluorescence intensity and fluorescence polarization of the cell population was detected for the first 100 min. However, at 100 min, a plateau in both fluorescence intensity and polarization was observed and was followed by a decrease in fluorescence polarization and a corresponding increase in fluorescence intensity. Western blot analyses revealed that the decrease in fluorescence polarization was due to proteolytic degradation of the probe within the cell. Using a combination of in vitro experiments and an additional fluorescent probe, it was determined that the initial increase in fluorescence polarization was due to movement of the probe through a pH gradient within the cell, suggestive of transport through the endocytic system. By combining fluorescence polarization, flow cytometry, and a unique fluorescent enhancement substrate, these studies represented a novel approach for monitoring intracellular trafficking and processing of proteins within macrophages.

Two-photon fluorescence lifetime imaging microscopy of macrophage-mediated antigen processing.

Two-photon fluorescence lifetime imaging microscopy was used noninvasively to monitor a fluorescent antigen during macrophage-mediated endocytosis, intracellular vacuolar encapsulation, and protease-dependent processing. Fluorescein-conjugated bovine serum albumin (FITC-BSA) served as the soluble exogenous antigen. As a relatively nonfluorescent probe in the native state, the antigen was designed to reflect sequential intracellular antigen processing events through time-dependent changes in fluorescence properties. Using two-photon lifetime imaging microscopy, antigen processing events were monitored continuously for several hours. During this time, the initial fluorescein fluorescence lifetime of 0.5 ns increased to approximately 3.0 ns. Control experiments using fluorescein conjugated poly-L-lysine and poly-D-lysine demonstrated that the increase in fluorescence parameters observed with FITC-BSA were due to intracellular proteolysis since addition of the inert D-isomer did not promote an increase in fluorescence lifetime or intensity. Comparisons of intravacuolar and extracellular FITC-dextran concentration suggested active localization of dextran in the vacuoles by the macrophage. In addition, the kinetics of degradation observed using two-photon microscopy were similar to results obtained on the flow cytometer, thus validating the use of flow cytometry for future studies.

Evidence for hapten recognition in receptor-mediated intracellular uptake of a hapten-protein conjugate by murine macrophage.

Fluorescein-derivatized bovine serum albumin (FITC-BSA) was used as an exogenous antigen and fluorescent probe to measure the kinetics of antigen uptake into the endocytic pathway of murine macrophage, J774, using flow cytometry. Results revealed dependency of the rate of antigen uptake on epitope density (moles FITC/mole BSA) implicating a role for FITC in the endocytosis of the derivatized antigen. In addition, inhibition of clathrin-coated pit formation in macrophage resulted in significantly reduced uptake of differentially labeled FITC BSA probes indicating receptor-mediated endocytosis via clathrin-coated pits. Fluoresceinamine (I) was found to inhibit the endocytic uptake of FITC-BSA at 10(-6) M. Determination of fractional receptor occupancies in macrophage upon binding different FITC BSA probes and calculation of the corresponding association rates (k(on)) for these binding events yielded values of 4.2+/-0.2 x 10(6)/M/min for FITC5BSA and 1.9+/-0.1 x 10(7)/M/min for FITC22BSA, respectively, at 37 degrees C. The five-fold difference in the rates of binding and endocytosis between the two probes was discussed on the basis of receptor cross-linking by a multivalent ligand (FITC22BSA), in contrast to monovalent ligand binding, on the cell surface that would lead to more rapid and efficient internalization of the FITC22BSA antigen.