Pulmonary Interstitial Macrophages: Isolation and Flow Cytometric Comparisons With Alveolar Macrophages and Blood Monocytes

Abstract

Pulmonary interstitial macrophages (IM) were isolated from rat lungs by an Fc gamma receptor-based affinity technique coupled with multiparameter flow cytometry. Single cell suspensions obtained by collagenase digestion of extensively perfused and lavaged lungs were applied to carpets of opsonized sheep red blood cells (SRBC-IgG) bound to plastic tissue culture flasks. At 0-4 degrees C, optimal binding of lung cells occurred within 60 min at plating densities of 1-2 X 10(6) lung cells/cm2 when the SRBC substrate was opsonized with 10 micrograms/ml anti-SRBC IgG. Nonadherent cells were removed by gently rinsing the plates and adherent cells were recovered by lysing the SRBC-IgG substrata. By light microscopy, the mixture of adherent cells was comprised of mononuclear cells (approximately 54%), many of which appeared to be macrophages, lymphocytes (approximately 20%), polymorphonuclear leukocytes (approximately 15%), plasma cells (approximately 8%), eosinophils (approximately 2%), and mast cells (approximately 0.5%). The cells which adhered to the SRBC-IgG monolayers were further resolved into subpopulations by multiparameter flow cytometry and sorted according to their electro-optical characteristics. One subpopulation appeared morphologically to be macrophages, and greater than 90% of these cells readily phagocytized SRBC-IgG in vitro. Peroxidase staining of this population was minimal, indicating that these cells were not blood monocytes (BM). Using a method by which alveolar macrophages (AM) were prelabeled with SRBC-IgG in situ, we demonstrated that alveolar macrophages constituted only approximately 5% of the total adherent cell population. We concluded from these observations that the macrophage population harvested in this manner were neither BM nor AM, but, rather, were harvested from the lung's interstitial compartment. Flow cytometric analyses indicated that the IM exhibited electro-optical characteristics intermediate between those of BM and AM, which is consistent with the concept of the lung's interstitium as a maturation compartment for the BM prior to migration into the alveolar compartment. However, the IM more closely resembled the BM than the AM, indicating that if the IM is in fact a precursor to the AM population, substantial maturation or differentiation must occur subsequent to its migration into the alveolar compartment. This isolation technique will be useful for harvesting highly purified IM for in vitro investigations.

Assessment of Nanobiotechnology-Targeted siRNA Designed to inhibit NF-kappaB Classical and Alternative Signaling in Breast Tumor Macrophages

“…The resulting ManNPs exhibited diameters of <40 nm (Figure 2), a size range that confers the additional advantage of exhibiting improved passive accumulation into tumors. 39,46 This approach extends previous work by Convertine et al describing polymers for siRNA delivery applications, which exhibited nonselective transfection activity driven by their cationic corona. 20 The diblock copolymers tested in this work were patterned after these polymers and provide a reliable benchmark against which the ManNPs can be reasonably compared.…”

“…SPMs and TAMs will be isolated from dissociated whole tissue cell suspensions with a 60 minute rapid adhesion protocol. 40,46 10 nM siRNA will be delivered to the cells for 18 hours using Lipofectamine® RNAiMAX transfection agent using the optimized protocol from Aim #1. Fluid motion will be created in the wells using a cell rocker, rocking at approximately 10 rocks per minute.…”

Novel flow‐cytometric method for separating cell types in differentiated F9 embryoid bodies

“…These embryoid bodies remained undifferentiated until they were induced to form an endodermal epithelium by culturing for 8 …”

“…In this report we describe differences in autofluorescence between cells in RA-induced embryoid bodies and demonstrate that cell types in differentiated embryoid bodies can be separated by flow cytometry according to these differences. Differences in cellular autofluorescence have been used previously to discriminate between various cell types, including alveolar macrophages from other accessory cells in the human lung and blood monocytes from alveolar macrophages (8,17). This sorting protocol offers the advantage over density-based separation that any number of parameters, such as cell size, cellular autofluorescence, and external and internal cellular morphology, can be used solely or in combination for the separation of cell types.…”

Cellular Defences of the Lung: Comparative Perspectives

“…There is, however, contrary evidence that PIMs represent a distinct population of cells with dedicated pulmonary inflammatory and immunoregulatory roles of defending the lung (e.g. Chandler et al, 1988;Dethloff and Lehnert, 1988;Lehnert, 1992;Prokhorova et al, 1994;Johansson et al, 1997;Zetterberg et al, 1998;Steinmüller et al, 2000). Although concerning inflammation and antimicrobial defense AMs exhibit greater functional repertoire related to and including increased chemotaxis, phagocytosis, cytotoxicity, and release of ROSs, PIMs express higher quantities of C3-receptor and intercellular adhesion molecule-1 and are more active in producing interleukins-1 and -6 (IL-1 and -6) and exhibit greater I-a antigen expression (Chandler and Brannen, 1990;Franke-Ullmann et al, 1996;Steinmüller et al, 2000).…”