Meryle J. Melnicoff

Fluorescent cell labeling for in vivo and in vitro cell tracking

Publisher Summary In cell biology, a valuable goal is to arrive at a better understanding of how specific cell types perform in vivo. This chapter discusses the molecular nature of intercellular recognition processes, an example of which is the lymphocyte T-cell receptor. While these receptors are important, they are functional only at close range. The ability to track the in vivo migration of labeled cells provides insight into differentiation processes in tissues, such as thymus or bone marrow. Cell-tracking technology is applied to tumor biology, to determine whether tumor-infiltrating lymphocytes migrate to metastatic sites. The ability to study cellular migration into atherosclerotic plaque would be of extreme value in the study of the pathophysiology of coronary disease. Information is obtained by studying the migration of cells into and out of an organ prior to and during the rejection process after organ transplants. The chapter describes the use of PKH2 cell linker kits. The label is an aliphatic fluorescent chromophore. The staining methodology allows for labeling in the lipids of the cell membrane rather than surface protein labeling.

Kinetics of changes in peritoneal cell populations following acute inflammation

The kinetics of macrophage (M phi) recruitment to the peritoneum following the induction of acute inflammation by thioglycollate broth (TG) was evaluated after prelabeling resident M phi with the fluorescent cell tracking dye, PKH-1. Most of the PKH-1-labeled resident M phi disappeared from the recoverable peritoneal cell population within the first hour after injection of TG. This disappearance coincided with the inflammatory influx of neutrophils (PMNs) and was sustained for at least 5 days after administration of TG, although the PMN number had returned to resident levels by this time. PKH-1-labeled peritoneal M phi were observed again in most animals at 7 days after injection of TG. The number of labeled M phi recovered at 7 days was approximately twice the number of resident peritoneal M phi in control animals which did not receive the TG broth. These additional M phi may include progeny of either the resident M phi or other local M phi precursors, such as omental M phi, which were labeled by the PKH-1 injection.

Maintenance of peritoneal macrophages in the steady state

Resident peritoneal macrophages (Mø) were labeled in situ by intraperitoneal (i.p.) injection of the green fluorescent cell tracking dye PKH‐1. After immunofluorescence staining with Mø specific monoclonal antibodies (Mabs) and phycoerythrin (PE) second antibody, the resident Mø were labeled with both the green dye and red Mab label, while recruited Mø were labeled only with the red Mab tag. These populations were distinguished by two‐color flow cytometry.

The role of T cell phenotypes in decreased lymphoproliferation of the elderly

A possible mechanism of the decreased mitogen-induced lymphoproliferation of the elderly is a shift in the relative numbers of T lymphocyte subpopulations. Results from studies examining such changes have been conflicting. In an effort to resolve this conflict, alterations in T cell subsets in the peripheral blood of a large number of relatively healthy, elderly subjects (149; mean age 84.6) were evaluated. Although there were several differences in percentages of subpopulations between elderly and young subjects, no significant differences in the absolute numbers of lymphocytes, monocytes, or T cell subsets between the groups were observed. In addition, there was no correlation between mitogen-induced lymphoproliferation and T cell subsets. Since the decreased mitogen response could reflect a difference in the ability of one T cell subset to proliferate, the phenotype of the T cells after stimulation with PHA was determined. Although the elderly demonstrated a decreased number of all T cell subsets after PHA stimulation compared to young subjects, the most pronounced decrease was in CD8+ cells. Further, young individuals demonstrated a significant increase in the percentage of CD8+ cells (p less than 0.001) after 72 hr of culture with PHA; elderly subjects showed no change in the percentage of T cell subsets. Although both groups had an increase in interleukin 2 receptor (IL2R), transferrin receptor (TfR), and MHC class II Ag (HLA-DR) positive cells after PHA stimulation, the mean percentage of TfR+ cells was significantly greater in the young than in the elderly (p less than 0.05). This decreased expression of TfR+ cells in the elderly was reflected by a decreased percentage of CD8+ cells expressing TfR. In addition, the percentages of CD8+ cells, CD8+ cells expressing TfR, and CD8+ cells expressing IL2R after activation correlated with PHA-induced proliferation. These results suggest that the impaired lymphoproliferative ability of elderly cells is not related to the proportions of T cell subsets present in peripheral blood. Rather, there appears to be some defect in the ability of all T cells, but especially of the T suppressor/cytotoxic cells, of the elderly to respond to PHA manifested by a decreased expression of transferrin receptors.

In Vivo Labeling of Resident Peritoneal Macrophages

A novel method for labeling resident peritoneal macrophages (MO) by injection of a dye into the peritoneal cavity is described. The dye, which fluoresces green, is selectively taken up by the resident Mø. Dye labeled cells can be further characterized by labeling of cell surface antigens with monoclonal antibodies (Mabs) and phycoerythrin conjugated second antibody. After such labeling with the Mabs F4/80 or Mac 1 the resident Mø were labeled by both the green dye and the red Mab markers, while recruited Mø or neutrophils were labeled with just the red Mab; the two populations of cells were readily distinguished by two‐color flow cytometry. This technique enabled identification of resident and recruited Mø in each animal without the use of radioisotopes, irradiation, or bone marrow ablation. Sufficient numbers of cells can be analyzed from each animal so that Individual animals could be evaluated.

Effects of inhaled ammonium sulfate on benzo [a] pyrene carcinogenesis

The effect of inhaled ammonium sulfate on benzo[a]pyrene carcinogenesis in the lungs of Syrian golden hamsters was studied. Exposure to ammonium sulfate at an airborne concentration 20 times average United States ambient levels resulted in a significant depression (p less than 0.05) of benzo[a]pyrene carcinogenesis in the first 6 mo of the study. However, at 2 yr, the termination of the study, there were no differences in cancer incidence between groups receiving benzo[a]pyrene and benzo[a]pyrene plus ammonium sulfate. In addition, at the concentration studied, inhaled ammonium sulfate did not significantly increase the incidence or severity of pneumonitis or pulmonary fibrosis in the hamster. However, this inhalation did increase the incidence of emphysema but not the severity. The decreased incidence of cancer during the first 6 mo of this study in animals receiving both benzo[a]pyrene and ammonium sulfate suggests that interaction between sulfate and benzo[a]pyrene does occur, but is insufficient to afford long-term protection against the development of cancer. No enhancement of carcinogenesis by benzo[a]pyrene occurs in the presence of inhaled sulfate.