Showing papers by "National Jewish Health published in 1992"

Mechanisms of lipopolysaccharide-induced neutrophil retention: Relative contributions of adhesive and cellular mechanical properties

Abstract: Intravascular LPS rapidly induces neutrophil sequestration in pulmonary capillaries by mechanisms that, although currently unknown, must take into account the size difference between the neutrophil and capillary diameter. To determine whether LPS alters neutrophil stiffness, and hence the ability of neutrophils to traverse capillaries, neutrophil passage through pulmonary capillaries was modeled by passage through filters with 6.5-microns pores. LPS increased retention in the pores in a concentration-dependent fashion that required the presence of heat-inactivated platelet-poor plasma, and was evident as early as 10 min after stimulation. The effect of LPS on the structural properties of the neutrophil was then studied. LPS induced f-actin reorganization in neutrophils in the presence of plasma. Disruption of actin organization and assembly with cytochalasin D completely inhibited early LPS-induced retention and attenuated retention at later timepoints, indicating that LPS-stimulated retention depends on filament organization. LPS-induced actin assembly and retention were abrogated by an antibody directed against CD14, a putative LPS receptor. CD18-dependent adherence of neutrophils contributed significantly to retention only at later timepoints with no significant contribution to retention at 20 min as determined by inhibition of adherence with the mAb 60.3. Morphometric assessment of neutrophil accumulation in the lungs of rabbits given 1 microgram LPS showed a marked increase in apparent neutrophil number, which was unaltered by antibodies to CD18, suggesting that mechanisms other than adhesion may account for accumulation in vivo. Direct measurements showed that neutrophil stiffness increased with exposure to LPS in a fashion similar to LPS-induced retention and actin organization. Pretreatment of neutrophils with cytochalasin D attenuated the increased stiffness. These data suggest that reorganization of filamentous-actin induced by LPS leads to cell stiffening and retention in capillary-sized pores. Although the organization of f-actin continues to be important in retention at later time points, adherence of cells also contributes significantly to cell retention. The changes in mechanical properties of the neutrophil may be important in the sequestration of neutrophils in pulmonary capillaries noted in endotoxemia.

Translocation of spectrin and protein kinase C to a cytoplasmic aggregate upon lymphocyte activation.

Abstract: We have previously reported that mammalian tissue lymphocytes exhibit significant heterogeneity with respect to the subcellular distribution of spectrin and that this phenomenon may result from a dynamic behavior of spectrin in response to activation signals. Here, we further characterize the involvement of spectrin in lymphocyte activation by examining its relationship with protein kinase C (PKC). PKC isoenzymes are a family of cytosolic kinases that translocate from the soluble to particulate fraction upon cell stimulation. It is reported here that activation of lymph node T cells through the antigen-specific receptor, or direct activation of PKC by phorbol esters, results in a striking increase in cells expressing a cytoplasmic aggregate of spectrin. Additionally, a concurrent increase in cells expressing aggregates of the beta II isozyme of PKC is observed. Immunofluorescence staining revealed that spectrin and PKC beta II are colocalized in untreated lymphocytes and that these two proteins are coincidently translocated to the same focal aggregate within the cytoplasm following stimulation. This redistribution of spectrin and PKC beta is blocked by pretreatment with calphostin C, a specific inhibitor of PKC. Solubility studies showed that there is an increase of both proteins in the detergent-insoluble fraction of lymphocytes upon activation, and immunoprecipitation studies indicated that the soluble form of these molecules may be associated directly or indirectly as part of a complex of proteins. These data indicate that the positioning of the spectrin-based cytoskeleton is sensitive to activation signals and may play a role in the function or positioning of PKC beta II.

Idiopathic Pulmonary Fibrosis: Quantitative Assessment of Lung Pathology: Comparison of a Semiquantitative and a Morphometric Histopathologic Scoring System

Abstract: The objective of this study was to determine the accuracy of a semiquantitative method of assessing the relative degree of cellularity and fibrosis compared with a morphometric analysis of specific histopathologic features in idiopathic pulmonary fibrosis (IPF). Morphometric analysis was performed on biopsy tissue from 20 patients. Morphometry showed that approximately 70% of parenchymal tissue was abnormal: 35% cellular consolidation and fibrosis, 20% honeycomb changes, 10% thick alveolar septa, and 5% small airways within abnormal parenchymal tissue. The prominent components of the abnormal parenchymal tissue were extracellular fibers, an abundance of interstitial cells, and epithelial cell hyperplasia. Correlation between four groupings of pathologic features, identified by a semiquantitative analysis, and each of the components of the parenchymal lesions showed significant correlations between (1) the fibrotic or reparative factor (alveolar wall metaplasia, fibrosis, honeycombing, and smooth muscle and vascular changes) and components of honeycomb lesions (extracellular fibers, interstitial cells, and epithelial injury and repair), and (2) the inflammatory and exudative factor and extracellular fibers in the lesions. These results support that the scoring system used by the pathology panel provides an accurate assessment of pathologic features useful in the assessment of the extent and severity of the histopathologic lesions of IPF.

Platelet-activating factor-mediated transmembrane signaling in human B lymphocytes is regulated through a pertussis- and cholera toxin-sensitive pathway.

Abstract: Platelet-activating factor (PAF) stimulates human B cells, resulting in elevation of intracellular calcium and the release of inositol phosphates. This signaling pathway is inhibited in the presence of pertussis (PT) or cholera toxin (CT). Preincubation of human B cells with either toxin, but not their inactive subunits, for 3 h blocked these PAF-induced responses in two B-lymphoblastoid cell lines. This effect was time dependent, with some inhibition noted at 30 min, but only after preincubation for 2-3 h was maximum inhibition achieved. This inhibitory activity was also dose dependent. The toxins blocked both PAF-induced transmembrane uptake of Ca2+ as well as release of Ca2+ from internal stores, and were selective in that activation events after cross-linking of surface IgM were not affected. Further, the toxins did not appear to act through elevation of intracellular levels of cAMP. These data, coupled with previous observations on the absence of heterologous desensitization between PAF and sIgM receptors, may delineate distinct signaling pathways in human B cells. This may reflect different roles for GTP-binding proteins in the activation of human B cells.

Ig V kappa family repertoire of plasma cells derived from autoimmune MRL mice.

Abstract: V kappa gene family usage was determined in the resident in vivo-activated plasma cells of individual diseased MRL mice by using in situ hybridization. In this way, the entire autoimmune repertoire could be analyzed. Autoantibody levels and extent of glomerulonephritis were also measured, so that the severity of disease could be assessed. It was found that V kappa expression was highly variable from mouse to mouse. Some animals displayed a V kappa family repertoire similar to mitogen-stimulated cells and consistent with the size of the families. These animals tended to have lower disease indices. Other animals, which had higher disease indices, displayed considerable over- or underutilization of individual V kappa families. However, no particular V kappa families were repeatedly biased in their expression, as was found at the VH level with J558. Importantly, in the 10% of animals that expressed VH J558 exclusively, four or more V kappa families were expressed and multiple antiself specificities were produced. The data are most consistent with a number of J558 genes being expanded in a variety of self-specificities. However, because only VH J558 is expressed in these sicker animals, nonspecific polyclonal activation is highly unlikely. These results underscore the continuing evolution of the autoimmune repertoire, with considerable diversity at early stages followed by a highly selected repertoire in which a potential role for nonspecific polyclonal activation is virtually excluded.

Pharmacokinetics of low-dose methotrexate in adult asthmatics.

Abstract: Several reports have been published on the disposition of methotrexate (MTX) when given in low dosages, but none in asthmatic patients. To address this matter, pharmacokinetic studies were performed in nine patients with severe, steroid-requiring asthma (ages 18-76 yrs) after the sixth weekly intramuscular dose of MTX. Theophylline pharmacokinetic studies were also performed at baseline prior to the start of MTX treatment and at the time of the MTX studies. Total systemic clearance of MTX, given as mean (SD), was 122.6 (25.1) ml/minute, volume of distribution at steady state 0.49 (0.2) L/kg, half-life 3.1 (0.3) hours, mean residence time 5.0 (0.6) hours, and renal clearance 89.1 (36.3) ml/minute. These values are similar to those previously reported for other patient populations receiving low-dose MTX. Eight of these patients were evaluated for changes in theophylline pharmacokinetics. Theophylline clearance at week 6 decreased an average of 19% from baseline and correlated inversely with total MTX clearance. Percentage change in theophylline clearance from baseline was inversely correlated with MTX nonrenal clearance, but was not statistically significant. This finding may indicate competition for clearance pathways between MTX and theophylline.