Isolation and characterization of resident macrophages from the smooth muscle layers of murine small intestine

Abstract

Macrophages within the murine tunica muscularis were isolated and cultured for physiological studies. Following dispersion, macrophages were identified by phagocytotic activity of fluorescein isothiocyanate (FITC)-dextran. Immediately following isolation, macrophages were rounded and possessed fluorescent granula but developed a ramified shape after 3-4 days in culture. Resident and cultured macrophages were immunopositive for F4/80 and I-Ad/I-Ed. Greater than 90% of F4/80 positive cultured cells were FITC-dextran positive. Macrophages had resting membrane potentials (RMP) of -33.3 +/- 1.5 mV after 1 day in culture, which increased to -53.9 +/- 4.4 mV after 3-4 days. The change in RMP was associated with the development of an inward rectifying K+ current, and a decrease in a voltage-dependent, inactivating outward current. After 3-4 days in culture the inflammatory mediated substances adenosine triphosphate (ATP), platelet-activating factor and bacterial lipopolysaccharide induced increases in cytoplasmic Ca2+ ([Ca2+]i). Forskolin suppressed the ATP-induced increase in [Ca2+]i. Macrophages exhibited oxidative bursts, measured by oxidation of dihydrorhodamine-123 to rhodamine-123. Oxidative bursts coincided with a reduction in intracellular pH. Macrophages expressed a proton conductance that may participate in pH maintenance during reactive oxygen production. These results suggest that resident macrophages in the intestine may play a role in the immunological protection of the gut.

Phagocytotic Activation of Muscularis Resident Macrophages Inhibits Smooth Muscle Contraction in Rat Ileum

“…In the murine bone marrow-derived macrophages, phagocytosable size of latex beads (1.1-µm) stimulated the release of PGE 2 , whereas these macrophages did not respond to nonphagocytosable particles (≥ 40-µm) [24]. In the present study, we demonstrated that the injected FITC-dextran was phagocytosed by muscularis resident macrophages in rat small intestine same as previous reports [15,20,26]. The confocal microscopic study for FITC-dextran uptake and immunohistochemical study based on the results of double staining of dextran and COX-2 protein suggest that FITC-dextran-positive resident macrophages became immunoreactive to COX-2 antibody (Figs.…”

“…concentration: Resident macrophages were isolated from the intestinal muscularis by standard collagenase treatment method as previously described [20]. Freshly isolated macrophages on glass cover slips were loaded with fura-PE3 by exposure to a HEPES buffer solution (in mM: NaCl 125.4, glucose 11.5, KCl 5.9, MgCl 2 1.2, HEPES 10, pH 7.4, either CaCl 2 1.5 or EGTA 0.5) containing 5 µM fura-PE3 acetoxymethyl ester with 0.01% cremophor EL for 30-40 min in a dark room.…”

“…On the other hand, PGE 2 which is released from peritoneal macrophages has been demonstrated to produce hyperalgesia [4] and increased vascular permeability [11]. We have previously reported that the injected dextrans, phagocytosed by resident macrophages reside in the intestinal muscle layer [20,26,28]. However, the possible changes in the macrophages and smooth muscle functions after the phagocytotic stimulation have not been elucidated.…”

Quantifying Ca2+ Current and Permeability in ATP-gated P2X7 Receptors

“…We circumvented this problem by measuring the fractional contribution of Ca 2ϩ to the total ATP-gated membrane current (i.e. the Pf %) using a true-to-life concentration of extracellular Ca 2ϩ and an appropriate membrane potential of Ϫ60 mV (90,91). We found that empirical measurements using recombinant receptors ranged from 3 to ϳ15% by comparison to the average Pf % of the P2X family as a whole (8.6 Ϯ 0.8%; red dotted line of Fig.…”

Kit‐like immunoreactivity in the zebrafish gastrointestinal tract reveals putative ICC

“…Antibodies specific for this protein label ICC within GI tissues in several species, including humans, and have been widely utilized to both determine ICC cellular morphology, and to characterize sub-populations of ICC located in distinct layers of the muscular wall of the GI tract (Ward and Sanders, 1992;Ward et al, 1994;Burns et al, 1997;Ozaki et al, 2004;Komuro, 1999). Several classes of ICC have been identified.…”