Dennis E. Doherty

One of the key features associated with programmed cell death in many tissues is the phagocytosis of apoptotic bodies by macrophages. Removal of apoptotic cells occurs before their lysis, indicating that these cells, during the development of apoptosis, express specific surface changes recognized by macrophages. We have compared the mechanisms by which four different macrophage populations recognize apoptotic cells. Murine macrophages elicited into the peritoneal cavity with either of two different phlogistic agents were able to phagocytose apoptotic cells. This phagocytosis was inhibited by phosphatidylserine (PS), regardless of the species (human or murine) or type (lymphocyte or neutrophil) of the apoptotic cell. In contrast, the murine bone marrow macrophage, like the human monocyte-derived macrophage, utilized the vitronectin receptor, an alpha v beta 3 integrin, for the removal of apoptotic cells, regardless of their species or type. That human macrophages are capable, under some circumstances, of recognizing PS on apoptotic cells was suggested by the observation that PS liposomes inhibited phagocytosis by phorbol ester-treated THP-1 cells. These results suggest that the mechanism by which apoptotic cells are recognized and phagocytosed by macrophages is determined by the subpopulation of macrophages studied.

Leukocytes within the circulation are in dynamic equilibrium with a marginated pool, thought to reside mainly within the pulmonary capillaries. The size discrepancy between the mean diameter of circulating leukocytes (6-8 microns) and that of the pulmonary capillaries (approximately 5.5 microns) forces the cells to deform in order to transit the capillary bed. Consequently, we investigated the hypothesis that the biophysical properties of cell size and deformability determined differential leukocyte retention in the lung. Comparison of the filtration properties of human neutrophils, lymphocytes, monocytes, platelets, and erythrocytes through polycarbonate filters (5-micron pore diameter) revealed that the largest leukocytes (neutrophils and monocytes) were retained to the greatest extent and the smaller cells (lymphocytes and platelets) the least. Undifferentiated HL-60 cells, of greater diameter than their differentiated counterparts, were also retained to a greater extent, confirming that cell size was one important determinant of retention in these model capillaries. However, compared with neutrophils, which are of similar diameter, monocytes were retained to a greater extent, suggesting that monocytes might be less deformable than neutrophils. To test this hypothesis, deformability was measured directly using the cell poker. Monocytes were found to be the stiffest, neutrophils the softest, and lymphocytes intermediate. Glutaraldehyde treatment of neutrophils markedly increased their stiffness and decreased their ability to transit the pores of the filters in vitro and the pulmonary microvasculature of rabbits without changing their adhesive properties or size. These observations support the hypothesis that biophysical properties of leukocytes (size and deformability) determine in part their ability to transit the pulmonary capillaries and may determine the magnitude of their marginated pools.

We have recently demonstrated that the combination of IL-2 and IL-4 blunts T cell responses to glucocorticoids in steroid resistant (SR) asthma by reducing glucocorticoid receptor (GCR)-binding affinity. Since immune activation appears to be involved in the acquisition of steroid resistance, we sought to identify whether other cytokines could also induce diminished GCR-binding affinity. In the current report, utilizing a [3H]dexamethasone radioligand-binding assay and Scatchard analysis, we found that IL-13, a cytokine with similar actions as IL-4, could induce diminished GCR binding-affinity (GCR Kd = 34.4 +/- 2.3 nM with IL-13 vs Kd = 8.8 +/- 0.7 nM for unstimulated control cells; p < 0.001) in PBMC from normal subjects. In contrast, PBMC incubated with IL-1, IL-3, IL-5, IL-7, IL-8, IL-12, or granulocyte-macrophage-CSF had no effect on GCR-binding affinity; and no additive effect to the decreased GCR-binding affinity was noted when IL-13 was cocultured with IL-2 or IL-4. The cell target of IL-13-induced GCR effects was studied and found to reside in the non-T cell population; specifically, the monocyte fraction. To determine the functional significance of the decreased GCR-binding affinity, monocytes were pretreated with and without IL-1 3 prior to stimulation with LPS and hydrocortisone. IL-13 pretreatment of monocytes significantly diminished (p = 0.005) the suppressive effects of hydrocortisone on LPS-induced IL-6 production. IL-13, by virtue of its ability to induce diminished GCR-binding affinity, may contribute to impaired GC responsiveness during inflammatory illnesses.