A novel system for investigating the ability of smooth muscle cells and fibroblasts to regulate adhesion of flowing leukocytes to endothelial cells.

Stromal cells may contribute to the inflammatory processes which lead to the recruitment of circulating leukocytes. Here, we describe a multicellular model in which chosen cellular elements of tissue can be cocultured with endothelial cells (EC). Cocultures can be incorporated into a novel parallel plate flow chamber to determine if stromal cells influence the patterns of leukocyte adhesion to the EC. As an example relevant to the pathology of atherosclerosis, EC were cultured with arterial smooth muscle cells (SMC) of the 'secretory' phenotype. EC and secretory SMC were cultured on the opposite faces of commercially available porous polyethylene terepthalate (PET) culture inserts, which fitted into a parallel plate flow chamber. Binding of flowing purified lymphocytes, labelled with the fluorochrome calcein-AM, to cocultured EC was assessed by fluorescence microscopy. Lymphocyte adhesion was negligible on unstimulated EC cultured alone or cocultured with SMC. However, when tumour necrosis factor-alpha (TNF) was added to cocultures, the EC supported greatly increased levels of lymphocyte adhesion compared to TNF-treated EC cultured alone. Additionally, cocultured EC responded to TNF at concentrations far below those at which EC cultured alone responded. This priming was specific in that skin fibroblasts cocultured with EC did not modify lymphocyte adhesion induced by TNF. Thus, we have developed a coculture model to determine the ability of tissue stromal cells to modify leukocyte recruitment. This may have wide applications in the study of the cellular pathology of inflammation by allowing the contribution of the local microenvironment to be assessed.

Migration of CD18-deficient neutrophils in vitro: evidence for a CD18-independent pathway induced by IL-8.

Neutrophils isolated from a child with severe leukocyte adhesion deficiency 1 (LAD1) had a complete absence of expression of the CD11/CD18 beta2 integrin family of adhesion molecules, and were shown to be deficient in the in vitro adhesion and migration properties. However, we found that interleukin-8 (IL8), a potent chemoattractant for neutrophils, and sputum sol phase induced these LAD1 neutrophils to migrate through an endothelial cell layer in vitro, and confirmed that this migration was CD18-independent. These findings add to evidence of CD18-independent mechanisms of neutrophil recruitment, in particular neutrophil infiltration into the lungs, where IL8 may be an important recruitment factor.

Chemokine and chemokine receptor interactions provide a mechanism for selective T cell recruitment to specific liver compartments within hepatitis C-infected liver.

The role played by chemokines in regulating the selective recruitment of lymphocytes to different tissue compartments in disease is poorly characterized. In hepatitis C infection, inflammation confined to portal areas is associated with a less aggressive course, whereas T cell infiltration of the liver parenchyma is associated with progressive liver injury and cirrhosis. We propose a mechanism to explain how lymphocytes are recruited to hepatic lobules during bursts of necroinflammatory activity in chronic hepatitis C infection. We report here that lymphocytes infiltrating hepatitis C-infected liver express high levels of the chemokine receptors CCR5 and CXCR3. However, whereas the CCR5 ligands macrophage inflammatory protein-1alpha and -1beta were largely confined to vessels within portal tracts, the CXCR3 ligands IFN-inducible protein-10 and monokine-induced by IFN-gamma were selectively up-regulated on sinusoidal endothelium. In vitro, human hepatic sinusoidal endothelial cells secreted IFN-inducible protein-10 and monokine-induced by IFN-gamma in response to stimulation with IFN-gamma in combination with either IL-1 or TNF-alpha. This suggests that intrahepatic Th1 cytokines drive the increased expression of IFN-inducible protein-10 and monokine-induced by IFN-gamma and thereby promote the continuing recruitment of CXCR3-expressing T cells into the hepatic lobule in chronic hepatitis C infection.

Promotion of leukocyte transendothelial cell migration by chemokines derived from human biliary epithelial cells in vitro.

Biliary epithelial cells are the focus of inflammatory damage in several liver diseases, including allograft rejection wherein intrahepatic bile ducts are infiltrated and damaged by T cells and neutrophils. Locally secreted chemotactic cytokines (chemokines) are important signals for leukocyte recruitment to an inflammatory site and include interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1), potent chemotactic agents for neutrophils and monocyte or T cells, respectively. In this study, we demonstrate that primary cultures of human biliary epithelial cells (BECs) express and secrete IL-8 and MCP-1, both of which are upregulated rapidly and markedly in response to the proinflammatory cytokines IL-1 and tumor necrosis factor-alpha. Interferon-gamma had a differential effect by reducing IL-8 secretion but stimulating MCP-1 secretion. BECs cocultured in transwell chambers below confluent monolayers of endothelial cells promoted the transendothelial migration of neutrophils, which was blocked by antibodies to CD18 or CD11b but only partially inhibited by blocking antibodies to IL-8. We conclude that human BECs produce and secrete potent, functional chemokines when stimulated by proinflammatory cytokines. The ability of BECs to secrete chemokines and thus to promote leukocyte infiltration into portal tracts seems likely to be an important cause of bile duct damage in such conditions as liver allograft rejection and may explain the involvement of intrahepatic bile ducts in a number of inflammatory liver diseases.

Cell cultures from bronchial subepithelial myofibroblasts enhance eosinophil survival in vitro.

Mechanisms of eosinophil accumulation and activation in the bronchial mucosa are crucial for the pathogenesis of asthma. The location of specialized fibroblasts, myofibroblasts, beneath the bronchial basement membrane and their proximity to infiltrating eosinophils potentially enable the myofibroblasts to modulate eosinophil survival and function in asthma. The aim of this study was to investigate the effects of bronchial myofibroblasts on eosinophil survival in vitro. Eosinophils from human peripheral blood were exposed to cell cultures from bronchial myofibroblasts and to myofibroblast-conditioned media. Eosinophil viability was assessed and granulocyte/macrophage colony-stimulating factor (GM-CSF) production was examined in co-culture supernatants and as messenger ribonucleic acid (mRNA) in myofibroblasts. Eosinophil survival was significantly increased and eosinophil apoptosis was inhibited by co-culture with myofibroblasts. Conditioned medium from tumour necrosis factor-alpha (TNF-alpha)-stimulated myofibroblasts also prolonged eosinophil survival. This effect could be blocked by GM-CSF antibody. GM-CSF mRNA and secretion from myofibroblasts were increased in co-cultures and by eosinophil-conditioned medium. Addition of antibodies to TNF-alpha and interleukin-1 alpha (IL-1 alpha) to co-cultures resulted in significant reduction both in eosinophil survival and GM-CSF levels. Blocking of fibronectin in the co-cultures did not affect the eosinophil survival enhancing activity. Prednisolone inhibited the eosinophil survival enhancing activity of the co-cultures by suppression of GM-CSF production. Soluble eosinophil-derived cytokines are involved in the interaction of eosinophils with myofibroblasts, which results in a tumour necrosis factor-alpha/interleukin-1 alpha mediated release of granulocyte/macrophage colony-stimulating factor from myofibroblasts. Bronchial myofibroblasts can, thereby, contribute to allergic inflammation by granulocyte/macrophage colony-stimulating factor-mediated inhibition of eosinophil apoptosis.

Selective eosinophil leukocyte recruitment by transendothelial migration and not by leukocyte-endothelial cell adhesion.

Eosinophil infiltration is the hallmark of allergic inflammatory events. However, the mechanisms governing the influx of eosinophils into the tissue at a site of an allergic reaction remains unclear. We have examined the interactions of eosinophils and neutrophils isolated from the same atopic donor with cultured human umbilical vein endothelial cell (EC) monolayers in the search for a mechanism for this selective eosinophil recruitment. First, the adherence of eosinophils and neutrophils to ECs stimulated with lipopolysaccharide, interleukin (IL)-1 alpha, and tumor necrosis factor-alpha were compared. Each mediator induced a similar dose-dependent enhancement of eosinophil adhesiveness for both eosinophils and neutrophils. Thus, although cytokine activation of ECs in the vasculature adjacent to an inflammatory site probably serves as an important focusing mechanism for the extravasation of inflammatory cells at this site, there does not appear to be any selective EC-dependent mechanism for eosinophil recruitment. Little or no effect on eosinophil and neutrophil adherence was observed with IL-3, IL-5, granulocyte/macrophage colony-stimulating factor, platelet-activating factor (PAF), leukotriene B4, or histamine. Second, the migration of eosinophils and neutrophils through an EC monolayer in response to chemoattractants was examined. PAF was found to selectively enhance eosinophil transendothelial migration at doses of 10(-7) to 10(-10) M, with optimal effect at 10(-8) M. This effect was gradient dependent and could be inhibited by WEB 2086, a specific PAF inhibitor. These results suggest that localized production of PAF may be a prime factor in the events leading to eosinophil accumulation at allergic inflammatory sites, and that selectivity for eosinophil recruitment occurs at the stage of transendothelial cell migration under the influence of cell-specific chemoattractants.

Different types of segmental sclerosing glomerular lesions in six experimental models of proteinuria.

From 133 to 615 glomeruli were examined in sections of kidneys from each of 60 animals, representing six rodent models of proteinuria. Particular attention was paid to the position of segmental lesions. Lewis rats given sheep anti-rat glomerular basement membrane antibodies had lesions almost exclusively at the glomerulo-tubular junction. Wistar rats on a diet of 24 per cent casein or with subtotal nephrectomy and a diet of 24 per cent soya had lesions mainly at the hilum. Wistar rats given bovine serum albumin had global lesions but virtually no segmental lesions. Wistar rats given puromycin aminonucleoside had lesions at the glomerulo-tubular junction and global mesangial abnormalities shortly after the treatment but later developed segmental lesions at all parts of the glomerulus. Untreated BUF/Mna rats had lesions at the glomerulo-tubular junction early in life but later had lesions at all parts of the glomerulus. Untreated NZB/NZW hybrid mice had various types of glomerulonephritis and also had lesions at the glomerulo-tubular junction. These findings showed that (1) segmental lesions at the glomerulo-tubular junction, or glomerular tip, occur in experimental animals, a fact not previously reported, and these tip changes are a common feature in several different models of proteinuria; (2) hilar segmental lesions are seen in conditions with hyperfiltration of protein; and (3) segmental lesions at various parts of the glomerulus are seen in some models of proteinuria and probably indicate late effects of random toxic damage to the glomerulus. Thus, there are at least three different types of segmental glomerular lesions in experimental animals--tip, hilar, and random--with different morphology and pathogenesis. It is likely that these findings can be extended to human renal diseases with segmental glomerular lesions. This will help to clarify the controversial and unsatisfactory term focal segmental glomerulosclerosis.