Extensive Mycobacterium bovis BCG infection of liver parenchymal cells in immunocompromised mice.

A histologic study was performed on the livers of wild-type (WT), severe combined immunodeficient (SCID), hydrocortisone acetate (HC)-treated WT, and HC-treated SCID mice infected intravenously with 10(5) CFU of Mycobacterium bovis BCG. It was found that infection progressed faster in SCID mice than in WT mice and that HC treatment caused exacerbation of infection in both types of mice. In all cases infection in the liver was confined to granulomas that were populated predominantly by macrophages. Higher levels of infection in HC-treated SCID mice, but not HC-treated WT mice, were associated with extensive infection and destruction of parenchymal cells at the margins of granulomas. The results indicate that in the absence of T-cell-mediated immunity and of HC-sensitive T-cell-independent defense mechanisms, macrophages are incapable of restricting BCG growth and of confining infection to their cytoplasm. Consequently, BCG bacilli are released into the extracellular environment, where they are ingested by neighboring parenchymal cells.

The cytoskeleton and cell volume regulation.

Although the precise mechanisms have yet to be elucidated, early events in osmotic signal transduction may involve the clustering of cell surface receptors, initiating downstream signaling events such as assembly of focal adhesion complexes, and activation of, e.g. Rho family GTPases, phospholipases, lipid kinases, and tyrosine- and serine/threonine protein kinases. In the present paper, we briefly review recent evidence regarding the possible relation between such signaling events, the F-actin cytoskeleton, and volume-regulatory membrane transporters, focusing primarily on our own work in Ehrlich ascites tumer cells (EATC). In EATC, cell shrinkage is associated with an increase, and cell swelling with a decrease in F-actin content, respectively. The role of the F-actin cytoskeleton in cell volume regulation in various cell types has largely been investigated using cytochalasins to disrupt F-actin and highly varying effects have been reported. Findings in EATC show that the effect of cytochalasin treatment cannot always be assumed to be F-actin depolymerization, and that, moreover, there is no well-defined correlation between effects of cytochalasins on F-actin content and their effects on F-actin organization and cell morphology. At a concentration verified to depolymerize F-actin, cytochalasin B (CB), but not cytochalasin D (CD), inhibited the regulatory volume decrease (RVD) and regulatory volume increase (RVI) processes in EATC. This suggests that the effect of CB is related to an effect other than F-actin depolymerization, possibly its F-actin severing activity.

Effect of cytochalasins on F-actin and morphology of Ehrlich ascites tumor cells.

Cytochalasins have been used extensively to probe the role of F-actin in different aspects of cellular function. Most of the data obtained are interpreted on the basis of the well-established depolymerizing effects of cytochalasins on F-actin preparations in vitro. However, some evidence indicates that, in intact cells, different cytochalasins can have varying effects on cell morphology and F-actin content and organization. To examine this problem in more detail, we analyzed the effects of cytochalasins on the cell morphology of and F-actin content and organization in Ehrlich ascites tumor (EAT) cells. After a 3-min exposure to 0.5 microM cytochalasin D, B, or E, F-actin content was equally reduced in all cases and this correlated with a reduction in the amount of cortical F-actin associated with the EAT cell membrane. However, only with CE was cell morphology markedly altered, with the appearance of numerous blebs. At 10 microM, blebbing was present in all conditions and the organization of cortical F-actin was disrupted. F-actin content, however, was not further reduced by this higher concentration and in CD it was identical to control levels. Exposure of EAT cells to similar concentrations of cheatoglobosin C, an analog of the cytochalasins that has little to no affinity for F-actin, resulted in a loss of F-actin content, a reduction in F-actin fluorescence, but no change in cell morphology, including a complete lack of bleb formation. Myosin II immunoreactivity, concentrated in the cortical cytoplasm colocalized with F-actin and in an area associated with the Golgi, was reduced by the high-dose cytochalasin. These results demonstrate that caution must be exercised in the use of cytochalasins to probe the role of F-actin in cellular function and that several parameters must be analyzed to obtain an accurate assessment of the effect of cytochalasin on the actin filament system.

Role of the F-actin cytoskeleton in the RVD and RVI processes in Ehrlich ascites tumor cells.

The role of the F-actin cytoskeleton in cell volume regulation was studied in Ehrlich ascites tumor cells, using a quantitative rhodamine-phalloidin assay, confocal laser scanning microscopy, and electronic cell sizing. A hypotonic challenge (160 mOsm) was associated with a decrease in cellular F-actin content at 1 and 3 min and a hypertonic challenge (600 mOsm) with an increase in cellular F-actin content at 1, 3, and 5 min, respectively, compared to isotonic (310 mOsm) control cells. Confocal visualization of F-actin in fixed, intact Ehrlich cells demonstrated that osmotic challenges mainly affect the F-actin in the cortical region of the cells, with no visible changes in F-actin in other cell regions. The possible role of the F-actin cytoskeleton in RVD was studied using 0. 5 microM cytochalasin B (CB), cytochalasin D (CD), or chaetoglobosin C (ChtC), a cytochalasin analog with little or no affinity for F-actin. Recovery of cell volume after hypotonic swelling was slower in cells pretreated for 3 min with 0.5 microM CB, but not in CD- and ChtC-treated cells, compared to osmotically swollen control cells. Moreover, the maximal cell volume after swelling was decreased in CB-treated, but not in CD- or Chtc-treated cells. Following a hypertonic challenge imposed using the RVD/RVI protocol, recovery from cell shrinkage was slower in CB-treated, but not in CD- or Chtc-treated cells, whereas the minimal cell volume after shrinkage was unaltered by either of these treatments. It is concluded that osmotic cell swelling and shrinkage elicit a decrease and an increase in the F-actin content in Ehrlich cells, respectively. The RVD and RVI processes are inhibited by 0.5 microM CB, but not by 0.5 microM CD, which is more specific for actin.