VprBP Is Required for Efficient Editing and Selection of Igκ+ B Cells, but Is Dispensable for Igλ+ and Marginal Zone B Cell Maturation and Selection.

B cell development past the pro-B cell stage in mice requires the Cul4-Roc1-DDB1 E3 ubiquitin ligase substrate recognition subunit VprBP. Enforced Bcl2 expression overcomes defects in distal VH-DJH and secondary Vκ-Jκ rearrangement associated with VprBP insufficiency in B cells and substantially rescues maturation of marginal zone and Igλ(+) B cells, but not Igκ(+) B cells. In this background, expression of a site-directed Igκ L chain transgene increases Igκ(+) B cell frequency, suggesting VprBP does not regulate L chain expression from a productively rearranged Igk allele. In site-directed anti-dsDNA H chain transgenic mice, loss of VprBP function in B cells impairs selection of Igκ editor L chains typically arising through secondary Igk rearrangement, but not selection of Igλ editor L chains. Both H and L chain site-directed transgenic mice show increased B cell anergy when VprBP is inactivated in B cells. Taken together, these data argue that VprBP is required for the efficient receptor editing and selection of Igκ(+) B cells, but is largely dispensable for Igλ(+) B cell development and selection, and that VprBP is necessary to rescue autoreactive B cells from anergy induction.

Fibronectin: functional character and role in alcoholic liver disease.

Fibronectins are adhesive glycoproteins that can be found in tissue matrices and circulating in various fluids of the body. The variable composition of fibronectin molecules facilitates a diversity of interactions with cell surface receptors that suggest a role for these proteins beyond the structural considerations of the extracellular matrix. These interactions implicate fibronectin in the regulation of mechanisms that also determine cell behavior and activity. The two major forms, plasma fibronectin (pFn) and cellular fibronectin (cFn), exist as balanced amounts under normal physiological conditions. However, during injury and/or disease, tissue and circulating levels of cFn become disproportionately elevated. The accumulating cFn, in addition to being a consequence of prolonged tissue damage, may in fact stimulate cellular events that promote further damage. In this review, we summarize what is known regarding such interactions between fibronectin and cells that may influence the biological response to injury. We elaborate on the effects of cFn in the liver, specifically under a condition of chronic alcohol-induced injury. Studies have revealed that chronic alcohol consumption stimulates excess production of cFn by sinusoidal endothelial cells and hepatic stellate cells while impairing its clearance by other cell types resulting in the build up of this glycoprotein throughout the liver and its consequent increased availability to influence cellular activity that could promote the development of alcoholic liver disease. We describe recent findings by our laboratory that support a plausible role for cFn in the promotion of liver injury under a condition of chronic alcohol abuse and the implications of cFn stimulation on the pathogenesis of alcoholic liver disease. These findings suggest an effect of cFn in regulating cell behavior in the alcohol-injured liver that is worth further characterizing not only to gain a more comprehensive understanding of the role this reactive glycoprotein plays in the progression of injury but also for the insight further studies could provide towards the development of novel therapies for alcoholic liver disease.

Cellular fibronectin stimulates hepatocytes to produce factors that promote alcohol-induced liver injury.

AIM: To examine the consequences of cellular fibronectin (cFn) accumulation during alcohol-induced injury, and investigate whether increased cFn could have an effect on hepatocytes (HCs) by producing factors that could contribute to alcohol-induced liver injury. METHODS: HCs were isolated from rats fed a control or ethanol liquid diet for four to six weeks. Exogenous cFn (up to 7.5 µg/mL) was added to cells cultured for 20 h, and viability (lactate dehydrogenase,LDH), apoptosis (caspase activity) and secretion of proinflammatory cytokines (tumor necrosis factor alpha, TNF-α and interleukin 6 IL-6), matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of metalloproteinases, TIMPs) was determined. Degradation of iodinated cFn was determined over a 3 h time period in the preparations. RESULTS: cFn degradation is impaired in HCs isolated from ethanol-fed animals, leading to its accumulation in the matrix. Addition of exogenous cFn did not affect viability of HCs from control or ethanol-fed animals, and apoptosis was affected only at the higher concentration. Secretion of MMPs, TIMPs, TNF-α and IL-6, however, was increased by exogenously added cFn, with HCs from ethanol-fed animals showing increased susceptibility compared to the controls. CONCLUSION: These results suggest that the elevated amounts of cFn observed in alcoholic liver injury can stimulate hepatocytes to produce factors which promote further tissue damage.

Ethanol feeding potentiates the pro-inflammatory response of Kupffer cells to cellular fibronectin.

BACKGROUND: Excessive alcohol consumption leads to the increased extracellular matrix deposition of cellular fibronectin (cFn) in the liver, which is also implicated as an initiating event in the fibrogenic process. We propose that cFn directly stimulates Kupffer cells (KCs), which are involved in the early response to tissue damage, to produce factors that enhance the progression of alcohol-induced liver injury toward inflammation and fibrosis. METHOD: KCs were isolated from rats fed a control or ethanol liquid diet for 4 to 6 weeks. The effect of exogenous cFn on KC viability and the secretion of the cytokines, TNF-α and IL-6, as well as of matrix remodeling factors, MMP-2 and TIMP-2, was determined after 20 hours of cell culture. RESULTS: For KCs from both control- and ethanol-fed rats, viability remained unaffected by treatment with cFn. TNF-α and IL-6 production were increased in KCs exposed to cFn, with cells treated with 1, 2.5, and 5 µg/ml cFn secreting significantly higher levels of both cytokines compared with untreated cells (p < 0.05). Chronic ethanol administration resulted in a significantly enhanced secretion of IL-6 by KCs regardless of treatment with cFn. When MMP-2 protein and activity levels were measured by western blot analysis and gelatin zymography, respectively, we found that cFn stimulated a dramatic increase in both cells from ethanol- and control-fed rats, with the KCs from ethanol animals being more responsive to cFn at higher concentrations (p < 0.05). Significantly higher levels of TIMP-2, which inhibits both the activation and activity of MMP-2, were secreted by KCs treated with 5 µg/ml cFn. Correspondingly, more pro-MMP-2 than active-MMP-2 was detected. CONCLUSION: Altogether, these results show that cFn stimulates KCs to produce factors that may enhance the promotion of tissue damage and that ethanol administration increases these responses.

Impaired receptor-mediated endocytosis: its role in alcohol-induced apoptosis.

Hepatocyte apoptosis, inflammation, and fibrosis are prominent features of liver disease in general and of alcoholic liver injury in particular. Although the link between these processes remains unclear, one universal characteristic of liver injury is the induction of hepatocellular damage, which results in the generation of apoptotic bodies. Work from our laboratory over the last several years has studied the effect of ethanol administration on the process of apoptosis and a role for altered endocytosis in alcoholic apoptosis. We initially focused our research on the hepatocyte by examining endocytosis using the asialoglycoprotein receptor (ASGP-R) pathway as a model and we identified multiple ethanol-induced impairments in receptor function. We also showed that uptake of apoptotic bodies is impaired in hepatocytes isolated from ethanol-fed animals compared to controls, and that this impairment is linked to altered ASGP-R function. Recent work from our laboratory is examining a link between ethanol-impaired ASGP-R function, apoptotic body accumulation, and inflammation in the liver. We are particularly interested in data showing that factors produced by Kupffer cells incubated with apoptotic bodies can lead to production of tumor necrosis factor-alpha and interleukin-6, and that this effect is exacerbated in the setting of alcohol administration. In addition, we have preliminary data showing that media from Kupffer cell cultures incubated with apoptotic bodies can induce hepatocyte killing. The goal of our future work is to show that inadequate removal of apoptotic cells, in part via altered receptor-mediated endocytosis, plays a role in the course of pathogenesis of alcoholic liver injury.