Comparative adhesion of human haemopoietic cell lines to extracellular matrix components, bone marrow stromal and endothelial cultures.

We used flow cytometry to characterize cell adhesion molecule expression of the human haemopoietic cell lines KG1a, K562, HL-60, NALM-6 and CEM. A 51chromium labelling assay was used to study the adhesion of these cell lines to extracellular matrix components and to bone marrow stromal and endothelial cultures. Both adhesion molecule expression and functional binding behaviour varied between cell lines. All five cell lines expressed the integrins alpha4beta1 and alpha5beta1 and all adhered to fibronectin. However, differences in intensity of expression of these integrins failed to correlate with extent of fibronectin adhesion. Inhibition experiments demonstrated that adhesion of KG1a to fibronectin was completely inhibited by divalent cation chelation and partially inhibited by RGDS peptides and chondroitinase ABC, suggesting that both alpha4beta1 and alpha5beta1 as well as CD44 were responsible for this interaction. Adhesion to bone marrow stromal and endothelial layers was superior to that to purified extracellular matrix components and was partially inhibited by divalent cation chelation. RGD peptides and anti-alpha4 monoclonal antibody also partially inhibited KG1a adhesion to bone marrow endothelium. Discordance between cell adhesion molecule expression and adhesive behaviour suggest that current phenotypic descriptions remain incomplete and reinforce the need for complementary functional binding studies.

Susceptibility of lung epithelium to neutrophil elastase: protection by native inhibitors.

The development of emphysema is thought to be due to an imbalance of proteases (especially neutrophil elastase [NE]) and antiproteases with loosening of the respiratory epithelium as an early event. We investigated the effect of NE on respiratory epithelial cell adherence in vitro , in the presence of varying concentrations and combinations of native inhibitors, alpha-1-proteinase inhibitor (PI) and secretory leukoprotease inhibitor (SLPI). SLPI was two to 12 times more effective than PI at preventing the effects of NE, especially when enzyme:inhibitor ratios were almost equivalent. Even when the concentration of SLPI was only 10% of the total (as in normal peripheral lung secretions), it gave greater protection than PI alone. This suggests that SLPI plays an important role in controlling neutrophil elastaseinduced inflammation and tissue damage.

Isolation and culture of endothelial cells from human bone marrow.

Adhesive interactions between haemopoietic progenitor cells and bone marrow sinusoidal endothelium are potentially important in the homing of these cells back to the extravascular compartment of the marrow to re-establish haemopoiesis following stem cell transplantation. A simple method for the isolation and culture of human bone marrow endothelial cells is described using bone marrow aspirates obtained from patients undergoing bone marrow harvests for autologous or syngeneic bone marrow transplantation. The method is based on the selective binding of the lectin Ulex europaeus agglutinin-1 (UEA-1) to endothelial cells. Magnetic Dynabeads coupled with UEA-1 were incubated with single cell suspensions of bone marrow following red cell lysis, and bound cells were isolated with a magnet. The isolated cells demonstrated positive immunofluorescence staining for von Willebrand factor. Cells were plated onto tissue culture flasks coated with extracellular matrix derived from human umbilical vein endothelial cells in an endothelial serum-free medium together with 5% fetal calf serum for 24 h. Cells were then cultured in endothelial serum-free growth medium supplemented with 5% fetal calf serum, endothelial cell growth supplement and heparin. After 2-4 weeks in culture, two morphologically different cell populations can be identified. One has a polygonal spindle-shaped morphology with a rapid growth rate, the other a rounded morphology and a slow growth rate. Both populations have a vesiculated cytoplasm. Positive immunostaining of the cells was demonstrated with a number of endothelial cell markers including von Willebrand factor, and antibodies to ICAM-1, VCAM-1, E-selectin, CD31 and BMA120. Weibel-Palade bodies were observed by electron microscopy. Culture of these cells will allow detailed in vitro studies of adhesion mechanisms in the homing of haemopoietic progenitor cells.

Immuno-, lectin-, and enzyme-histochemical characterization of human bone marrow endothelium.

"Homing" of hematopoietic progenitor cells to the bone marrow occurs during the clinical practice of bone marrow transplantation. Its mechanism is unknown, although adhesive interactions between hematopoietic cells and sinusoidal endothelium in the bone marrow may be implicated. Studies of human bone marrow endothelial cells have previously been limited by the lack of markers for these cells. In this report, we describe positive staining of bone marrow endothelial cells from human bone marrow trephine biopsies with antibody to factor VIII-related antigen (FVIIIR-Ag) (Dako, High Wycombe, UK), the plant lectin Ulex europaeus agglutinin-I (UEA-I), and two mouse monoclonal antibodies, BMA120 and QBEND/10. In addition, alkaline phosphatase could be demonstrated in the majority of marrow endothelial cells using a novel enzyme histochemical technique. These studies defined the marker profile of human marrow endothelium. The results of this study will facilitate the isolation and culture of human marrow endothelial cells for in vitro studies of their roles in hematopoietic stem cell homing.

Biochemical and cellular mechanisms of pulmonary fibrosis.

This review summarizes the manner in which a variety of agents may induce fibrogenic reactions in the lung. The extent of reaction is dependent on dose, time scale of exposure, and chemical reactivity. The regime of multiple dosing with chemicals or gases with recovery periods is important in disease progression. The means by which biochemists and histopathologists assess fibrosis, the advantages and disadvantages of each of the methods as related to subjectivity, quantitation, and speed of analysis are compared. The mechanisms which control the step from fibrogenesis (a potentially reversible reaction) to fibrosis (irreversible) may be linked to the maturation of collagen, calcification, or the formation of cross-linked protein masses. Attention is given to hydroxylysine cross-links in newly formed "fibrotic" collagen but focusses on gamma-glutamyl-epsilon-lysyl cross-links formed by calcium-dependent transglutaminases. It is suggested that these enzymes, released by replacement epithelial cells, could be responsible for the formation of stabilized protein masses in the lung, thus contributing to a progressive fibrosis.

Interactions between paraquat, endogenous lung amines' antioxidants and isolated mouse Clara cells.

The ability of paraquat to damage mouse lung Clara cells in the presence and absence of herbicide inhibitors is investigated using a cell culture system. Clara cell damage is assessed on the loss of nitroblue tetrazolium reductase activity and the inability to attach and spread on an extracellular matrix. Endogenous amines such as putrescine and spermidine reduce paraquat-induced damage at low concentrations indicating that they compete for the same cell surface receptor as paraquat and thus potentially block the accumulation of the herbicide. The efficacy of 10 microM exogenous putrescine as a protectant is reduced the longer the time before it is added to the cultures. Clara cells contain high levels of NADPH-dependent P-450 reductase which is required to redox cycle the paraquat and generate reactive oxygen radicals. Compounds with antioxidant properties are examined for their ability to reduce the Clara cell damage. Cystamine, the disulphide form of the naturally occurring thiol, cysteamine, and taurine, a metabolite of cystamine, both of which are accumulated in the lung, do not reduce paraquat-induced Clara cell damage. Another antioxidant, alpha-tocopherol is also ineffective but reduced glutathione (GSH), present in high quantities (3.2 mM) in clara cells, could reduce damage to the cultured cells. Cysteine, a precursor of GSH, can also prevent Clara cell damage when the concentration of paraquat is low.

The histopathology of rat lung following exposure to zinc oxide/hexachloroethane smoke or installation with zinc chloride followed by treatment with 70% oxygen.

The effects of inhaled zinc oxide/hexachloroethane smoke (11,580 mg x min/m3) and intratracheally instilled zinc chloride (2.5 mg/kg body weight) have been studied in rat lung. The effects of subsequent treatment with 70% oxygen have been studied after both procedures. Both the inhalation of the smoke and instillation of zinc chloride produced similar effects that included pulmonary edema, alveolitis and, at a later stage, some fibrosis. After zinc chloride instillation, the pathological changes largely spared the periphery of the lung, while following smoke inhalation they were more diffuse. Subsequent oxygen administration had little effect on the development or progression of the pathological changes.

The biochemical and pathological changes produced by the intratracheal instillation of certain components of zinc-hexachloroethane smoke.

Zinc chloride which is formed by igniting a mixture of zinc oxide and hexachloroethane in the production of white smokes has been shown to produce oedema when given to rats as a single instillation. The oedematous reaction, as assessed by histopathology and measurements of alveolar surface protein in lavage fluid, is variable, dose-dependent, and maximal at 3 days but at sub-lethal doses it regresses after 7 days. The parent compound, zinc oxide, does not produce these effects. In some animals there is evidence of a fibrogenic response at 7 days post-exposure although it is currently unknown whether or not this effect is progressive.