Publications

Infection of inbred mice with Leishmania major remains the best model of human infection with visceralizing Leishmania that cause kala-azar. Immunologic investigations have correlated the outcome of disease with expansion of different subsets of CD4+ cells, designated Th1 and Th2. Although the capacity of fixed effector Th1 and Th2 populations to mediate the diverse outcomes of disease through the release of soluble cytokines, particularly IFN-gamma and IL-4, has been demonstrated, the mechanisms by which these subsets become established during infection have not been delineated. This review focuses on known features of CD4+ differentiation using other experimental models, and proposes that genetic susceptibility to Leishmania can occur if the host has a Th2 precursor cell in the memory configuration prior to the time of exposure to organisms, perhaps in response to cross-reactive self-peptides. The hypothesis can explain a number of puzzling observations in both murine and human disease due to these organisms and makes several predictions amenable to experimental testing.

The development of techniques for isolating hepatic lipocytes (Ito, stellate or fat-storing cells) from rodents has been instrumental in defining their role in hepatic vitamin A storage and fibrogenesis. In this study, we developed a method for the purification of lipocytes and Kupffer cell from wedge sections of normal human liver and examined their properties in primary culture. Sections of donor liver (400 to 600 gm) harvested but not used for transplantation were perfused in situ with University of Wisconsin solution and used for lipocyte isolation within 48 hr. Cells were isolated by catheter perfusion of the wedge through several large vessels with L-15 salts, Pronase and collagenase, followed by Larex density gradient centrifugation. Lipocytes were plated on either uncoated plastic or a basement membrane-like gel. Lipocyte and Kupffer cell yields were 2.3 +/- 0.6 x 10(5) and 8.6 +/- 1.4 x 10(5) cells, respectively, per gram of liver (n = 5). Lipocyte purity was 91% as assessed by vitamin A autofluorescence, and Kupffer cell purity was 83% as determined by uptake of fluorescinated staphylococci. Lipocytes cultured on the plastic spread within 48 to 72 hr, displaying slightly more heterogeneous retinoid droplet size than comparable rat cells; on a basement-membrane gel, the cells remained aggregated and spherical with occasional spindlelike extensions. Lipocytes on plastic expressed procollagens I and III, collagen IV and laminin by immunocytochemistry, and types I, III and IV procollagen messenger RNAs by RNAse protection. Northern blot and polymerase chain reaction, respectively. Transmission electron microscopy of lipocytes at 7 days demonstrated a prominent rough endoplasmic reticulum and contractile filaments. Scanning electron microscopy revealed a smooth cell surface with perinuclear droplets beneath the cell membrane. With continued primary culture on plastic (more than 7 days), cells appeared "activated" (i.e., increased spreading and diminished retinoid droplets) and began proliferating as assessed by nuclear autoradiography and [3H]thymidine incorporation. Kupffer cells observed by scanning electron microscopy in early primary culture displayed prominent membrane ruffling and lamellipodia. In summary, we have established a reproducible method for the isolation and primary culture of human lipocytes and Kupffer cells.

Lipid A, the active component of bacterial endotoxin, stimulates multiple cell types, including glomerular mesangial cells (MC), and yet the molecular mechanisms of cell activation remain unclear. Lipid A, in its monosaccharyl form, structurally resembles the biologically active lipid phosphatidic acid (PA). Given this, it was postulated that lipid A activates cells by acting as a structural and functional mimetic of PA. Lipid A was found to specifically stimulate an MC lyso-PA acyl transferase activity, leading to enhanced synthesis of sn-2-unsaturated forms of PA. Sn-2-unsaturated PA itself, in contrast to sn-2-saturated PA, also stimulated the lyso-PA acyl transferase activity, a positive feedback feature previously noted with lyso-lecithin acyl transferase. Structure-function correlations demonstrated that the phosphate moieties in both PA and lipid A were necessary to feedback stimulation of lyso-PA acyl transferase (AT), as dephosphorylated lipid A and 2-unsaturated 1,2-sn-diacylglycerol had no stimulatory effect on lyso-PA AT. The biologic relevance of the lipid A and PA-mediated increases in lyso-PA acyl transferase activity was shown, whereby limited exposure to these lipids rapidly induced identical MC morphologic and functional alterations characteristic of cellular activation. By mimicking the stimulatory action of PA, per se, on lyso-PA acyl transferase activity, lipid A may initiate a positive feedback cycle of acylation, yielding increased amounts of PA enriched in unsaturated fatty acids. This newly synthesized PA may subsequently act as the proximal mediator of cellular activation.

Hormones inhibit synthesis of adenosine 3',5'-monophosphate (cAMP) in most cells via receptors coupled to pertussis toxin (PTX)-sensitive guanine nucleotide-binding (G) proteins. Mutationally activated alpha subunits of Gi2 (alpha i2) constitutively inhibit cAMP accumulation when transfected into cells. Cells have now been transfected with mutant alpha subunits of four other G proteins--Gz, a PTX-insensitive G protein of unknown function, and Gi1, Gi3, and G(o), which are PTX-sensitive. Mutant alpha z, alpha i1, and alpha i3 inhibited cAMP accumulation but alpha o did not. Moreover, expression of wild-type alpha z produced cells in which PTX did not block hormonal inhibition of cAMP accumulation. Thus, Gz can trigger an effector pathway in response to hormone receptors that ordinarily interact with PTX-sensitive Gi proteins.