Influence of immune complexes on macrophage membrane fluidity: a nanosecond fluorescence antisotropy study

Abstract

Time-resolved fluorescence anisotropy (TRFA) and steady-state anisotropy measurements and fluorescence intensification microscopic observations were made on RAW264 macrophages labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) or 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Microscopic analysis revealed that the fluorescent probe DPH was found in association with plasma membranes and small vesicles. Macrophages treated with immune complexes could not be distinguished from untreated cells, indicating that the same membrane compartments were labeled. The probe TMA-DPH was exclusively localized to the plasma membrane. Steady-state anisotropy measurements indicated that in vitro culture conditions did not significantly affect membrane fluidity. TRFA measurements were conducted to determine the physical properties of macrophage membranes during immune recognition and endocytosis. Data were analyzed by iterative deconvolution to yield phi, the rotational correlation time, and r infinity, the limiting anisotropy. These parameters may be interpreted as the "fluidity" and order parameter of the membrane environment, respectively. Typical values for untreated macrophages were phi = 7.8 ns and r infinity = 0.12. Binding and endocytosis of immune complexes prepared in 4-fold antigen excess increase these values to phi = 22.1 ns and r infinity = 0.15. However, receptor-independent phagocytosis of latex beads decreases these values to phi = 2.2 ns and r infinity = 0.10. Addition of catalase before, but not after, immune complex incubation with cells diminishes the effect upon membrane structure, suggesting that H2O2 participates in fluidity changes. Pretreatment of macrophages with the membrane-impermeable sulfhydryl blocker p-(chloromercuri)benzenesulfonic acid also diminished these effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison between flow cytometry and fluorometry for the kinetic measurement of membrane fluidity parameters

“…However, the anisotropy values of the positively charged TMA-DPH did not change appreciably (Figs. lD, 2A), suggesting that it remained in the HeLa plasma membrane, in agreement with the observations of Petty et al (11) made with macrophages and our visual observations. Numerous investigators have commented on the difflculty of visualizing the rapidly bleached fluorescence of cells stained with membrane probes.…”

“…However, their method used a prior fixation with glutaraldehyde, which might compromise the results. Petty et al (11) presented photographs of recorded video images of macrophages stained for 30 min with 0.5 pM TMA-DPH and DPH where TMA-DPH appeared to be exclusively on the cell surface (presumably the plasma membrane) and DPH appeared to be mostly on the surface, but also inside the cell.…”

“…A positively charged derivative, TMA-DPH, 1-[C(trimethylamino) phenyl]-6-phenyl-1,3,5-hexatriene, would be expected to penetrate only the outer hemileaflet of the membrane with the charged moiety remaining anchored at the cell surface (7). Potentially useful are a series of AS (anthroyloxystearate) probes with the fluorophore at different positions on the fatty acid chain [n-(9:anthroyloxy) fatty acids (where n = 2, 6,9,11,1511. The negatively charged AS series would be expected to penetrate the lipid bilayer with the fluorophore at different positions (17).…”

Plasma membrane fluidity gradients of human peripheral blood leukocytes

“…In recent animal cell studies, the binding of immune complexes (Petty et al, 1987) and activation with BCG (Somers et al, 1987) increased the plasma membrane fluidity of macrophages. Increased membrane fluidity of animal lymphocytes was observed after treatment with the lectin, Concanavalin A (Pajor et al, 19861, interleukin 2 (Utsunomiya et al, 19861, therapeutic doses of cis platinum drugs (Simpkins et al, 1986), and in magnesium deficiency (Gunther et al, 1984).…”

Optical microscopy of antibody‐dependent phagocytosis and lysis of erythrocytes by living normal and chronic granulomatous disease neutrophils: A role of superoxide anions in extra‐ and intra‐cellular lysis

“…Human neutrophils play a key role in host defense against bacterial, fungal, and viral invasion. Upon appropriate plasma membrane stimulation, a cascade of events takes place that includes increases in phospholipid turnover (Serhan e t al., 19821, increases in intracellular calcium (Takeshige et al, 1980;Mottola and Romeo, 1982), changes in membrane fluidity B e r l i n and Fera, 1977;Ingraham et al, 1981;Petty et al, 1987a), increased monovalent cation fluxes (Simchowitz et al, 19821, release of arachidonate metabolites (Walsh et al, 1981) and lysosomal enzymes (Goldstein and Weissmann, 1979), and the production of superoxide anions 0 1988 ALAN R LISS. INC and hydrogen peroxide (Klebanoff, 1980).…”