Ursodeoxycholic acid reduces CpG-induced IgM production in patients with primary biliary cirrhosis.

AIM: Ursodeoxycholic acid (UDCA) treatment reduces IgM serum levels in patients with primary biliary cirrhosis (PBC) without affecting serum antimitochondrial antibody (AMA) titers. We previously reported that PBC-associated hyper-IgM is secondary to a disease-specific hyperproduction following bacterial stimulation by B cells. METHODS: We isolated peripheral blood mononuclear cells (PBMC) from patients with PBC and controls and evaluated whether bacterial CpG challenge in the presence of UDCA at concentrations consistent with those achieved in treated patients led to changes in total IgM, IgG-AMA, and IgM-AMA production. Further, p65 phosphorylation and CD38 cell expression were analyzed as measures of activation of the NF-kB signaling pathway and B cell subsets, respectively. RESULTS: UDCA significantly reduced CpG-induced total IgM and IgM-AMA production, but had no impact on IgG-AMA production. UDCA also significantly reduced the activation ofnaïve and IgM memory, but not IgG memory, B cells, as represented by CD38 expression levels. Further, p65 phosphorylation was significantly reduced in the presence of UDCA. CONCLUSION: UDCA reduces total and IgM-AMA production in PBMC from patients with PBC by downregulating B cell activation and NF-kB signaling. These data ultimately suggest novel mechanisms of action for UDCA in chronic autoimmune cholestasis.

Hepatic IL-17 responses in human and murine primary biliary cirrhosis.

The emergence of new regulatory and pro-inflammatory immune cell subsets and cytokines dictates the need to re-examine the role of these subsets in various diseases involving the immune system. IL-17 has been recently identified as a key cytokine involved in numerous autoimmune processes. However, its role in liver autoimmune diseases remains unclear. Primary biliary cirrhosis (PBC) is characterized histologically by autoreactive CD4 and CD8 T cells surrounding damaged bile ducts. CD4(+) T cells are a major source of IL-17, which compose a distinct T helper subset (Th17). Thus we set out to determine the role of IL-17 in both human and a murine model of PBC in a liver-targeted manner. Our data demonstrate an increase in the frequency of IL-17(+) lymphocytic infiltration in liver tissues from PBC patients and those with other liver dysfunctions as compared to healthy livers. IL-2 receptor alpha knockout mice, a recently identified murine model of human PBC, also demonstrate marked aggregations of IL-17-positive cells within portal tracts and increased frequencies of Th17 cells in the liver compared to the periphery. Interestingly, CD4(+) T cells from livers of normal C57BL/6J mice also secreted higher levels of IL-17 relative to those from spleens, indicating a preferential induction of Th17 cells in liver tissues. Importantly, C57BL/6J cocultures of splenic CD4(+) T cells and liver non-parenchymal cells increased IL-17 production approximately 10-fold compared to T cells alone, suggesting a role of the liver microenvironment in Th17 induction in cases of liver autoimmunity and other liver inflammatory diseases.

Differential mechanisms in the pathogenesis of autoimmune cholangitis versus inflammatory bowel disease in interleukin-2Ralpha(-/-) mice.

Interleukin-2 (IL-2) receptor alpha knockout (IL-2Ralpha(-/-)) mice have a deficiency of CD25 and a corresponding functional defect in T regulatory cells (Tregs). These mice spontaneously develop portal inflammation with biliary ductular damage and colitis with features similar to human inflammatory bowel disease with T cell infiltrates in both the liver and colon. In humans, inflammatory bowel disease may be accompanied by primary sclerosing cholangitis (PSC), but seldom primary biliary cirrhosis (PBC). We hypothesized that the effector mechanism responsible for T cell infiltrates would differ for colon versus liver. To address this thesis, we developed three colonies of double-knockout mice including IL-2Ralpha(-/-) CD4(-/-), IL-2Ralpha(-/-) CD8(-/-), and IL-2Ralpha(-/-) T cell receptor (TCR)-beta(-/-). Tissue immunopathology, body weight, and serum levels of cytokines, immunoglobulins, and anti-mitochondrial antibodies (AMA) were assayed at 3 months of age. Relative to IL-2Ralpha(-/-) mice, IL-2Ralpha(-/-) CD4(-/-) mice had increased biliary ductular damage but reduced inflammation in the colon. In contrast, IL-2Ralpha(-/-) CD8(-/-) mice had increased colon inflammation but markedly attenuated biliary ductular damage. Both IL-2Ralpha(-/-) CD4(-/-) and IL-2Ralpha(-/-) CD8(-/-) mice demonstrated elevated serum levels of tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), interleukin-12p40 (IL-12p40), and interleukin-2 (IL-2) compared with C57BL/6J controls, but only IL-2Ralpha(-/-) CD8(-/-) mice had increased serum levels of immunoglobulin A (IgA), AMA and interleukin-17 (IL-17). Finally, and of importance, IL-2Ralpha(-/-) TCR-beta(-/-) mice had abrogation of liver and colon pathological conditions and lacked AMA. In conclusion, on loss of Treg function in mice, CD8 T cells mediate biliary ductular damage whereas CD4 T cells mediate induction of colon-specific autoimmunity.

Tyrosine sulfation is prevalent in human chemokine receptors important in lung disease.

Post-translational sulfation of tyrosines affects the affinity and binding of at least some chemokine receptors to their ligand(s) and has been hypothesized to be a feature in all chemokine receptors. This binding initiates downstream signaling cascades. By this mechanism, tyrosine sulfation can influence the cells involved in acute and chronic events of cellular immunity. These events include leukocyte trafficking and airway inflammation important in asthma and chronic obstructive pulmonary disease (COPD). We are using computational methods to convert the poorly defined hypothesis of more widespread sulfation of chemokine receptors to more specific assessments of how closely the sequence environment of each tyrosine residue resembles the sequence environment of tyrosine residues proven to be sulfated. Thus, we provide specific and readily tested hypotheses about the tyrosine residues in all of the chemokine receptors. Tyrosine sulfation was predicted with high scores in the N-terminus domain of 13 out of 18 human chemokine receptor proteins using a position-specific scoring matrix, which was determined to be 94.2% accurate based on Receiver Operating Characteristic analysis. The remaining chemokine receptors have sites exhibiting features of tyrosine sulfation. These putative sites demonstrate clustering in a manner consistent with known tyrosine sulfation sites and conservation both within the chemokine receptor family and across mammalian species. Human chemokine receptors important in asthma and COPD, such as CXCR1, CXCR2, CXCR3, CXCR4, CCR1, CCR2, CCR3, CCR4, CCR5, and CCR8, contain at least one known or predicted tyrosine sulfation site. Recognition that tyrosine sulfation is found in most clinically relevant chemokine receptors could help the development of specific receptor-ligand antagonists to modulate events important in airway diseases.

The current immune function of hepatic dendritic cells.

While only a small percentage of the liver as dendritic cells, they play a major role in the regulation of liver immunity. Four major types of dendritic cell subsets include myeloid CD8alpha(-)B220(-), lymphoid CD8alpha(+)B220(-), plasmacytoid CD8alpha(-)B220(+), and natural killer dendritic cell with CD8alpha(-)B220(-)NK1.1(+) phenotype. Although these subsets have slightly different characteristics, they are all poor naïve T cell stimulators. In exchange for their reduced capacity for allostimulation, hepatic DCs are equipped with an enhanced ability to secrete cytokines in response to TLR stimulation. In addition, they have increased level of phagocytosis. Both of these traits suggest hepatic DC as part of the innate immune system. With such a high rate of exposure to the dietary and commensal antigens, it is important for the hepatic DCs to have an enhanced innate response while maintaining a tolerogenic state to avoid chronic inflammation. Only upon secondary infectivity does the hepatic DC activate memory T cells for rapid eradication of recurring pathogen. On the other hand, overly tolerogenic characteristics of hepatic DC may be responsible for the increase prevalence of autoimmunity or liver malignancies.

Autoimmunity and tyrosine sulfation.

Homeostasis of the immune system is achieved through refined regulation and communication between immunologically relevant receptor and their cognate ligands amongst mononuclear cells during ontogeny and day to day immune responses. An aberrance in not only the kinetics of receptor expression but also the relative diversity of expression alter these events. More importantly, improper modulation of ligand binding affinity can be a triggering event that results in autoimmunity. As one of the most common post-translational modifications, tyrosine sulfation possesses the ability to regulate mononuclear cell function at various stages of the immune response. For example, removal of sulfated tyrosine residues consistently decreases the binding affinity of the ligand to its corresponding receptor as exemplified by studies of several tyrosine sulfated proteins such as PSGL-1, CD44v5, CCR5, and CXCR4, all known to play a role in a variety of autoimmune diseases. This review defines possible roles that tyrosine sulfated proteins may play in the pathogenesis of autoimmune diseases.

Chronic aeroallergen during infancy enhances eotaxin-3 expression in airway epithelium and nerves.

We have documented that exposure of rhesus monkeys to house dust mite aeroallergen during postnatal development resulted in significant recruitment of eosinophils into the airway mucosa (Clin Exp Allergy 33:1686-1694, 2003). Because eosinophils were not uniformly distributed throughout the five conducting airway generations examined, we speculated that trafficking within anatomic microenvironments of the lung is mediated by differential chemokine expression. To address this question, we used quantitative real-time RT-PCR to evaluate the related eosinophilic chemokines, eotaxin (CCL11), eotaxin-2 (CCL24), and eotaxin-3 (CCL26) within isolated airways of infant monkey lung. Overall, chemokine mRNA expression levels in house dust mite-exposed airways were as follows: eotaxin-3 > eotaxin > eotaxin-2. Immunofluorescence staining for eotaxin-3 and CC chemokine receptor 3 (CCR3) showed positive cells within epithelium and peripherally located nerve fiber bundles of the airway wall. Epithelial volume of eotaxin-3 within the trachea correlated with epithelial volume of major basic protein. CCR3+ and MHC Class II+ dendritic cells, but not eosinophils or mast cells, co-localized within eotaxin-3+ nerve fiber bundles. We conclude that localized expression of eotaxin-3 plays an important role in the recruitment of diverse CCR3+ cell populations to different anatomic microenvironments within the infant airway in response to chronic allergen exposure.

Prediction of tyrosine sulfation sites in animal viruses.

Post-translational modification of proteins by tyrosine sulfation enhances the affinity of extracellular ligand-receptor interactions important in the immune response and other biological processes in animals. For example, sulfated tyrosines in polyomavirus and varicella-zoster virus may help modulate host cell recognition and facilitate viral attachment and entry. Using a Position-Specific-Scoring-Matrix with an accuracy of 96.43%, we analyzed the possibility of tyrosine sulfation in all 1517 animal viruses available in the Swiss-Prot database. From a total of 97,729 tyrosines, we predicted 5091 sulfated tyrosine sites from 1024 viruses. Our site predictions in hemagglutinin of influenza A, VP4 of rotavirus, and US28 of cytomegalovirus strongly suggest an important link between tyrosine sulfation and viral disease mechanisms. In each of these three viral proteins, we observed highly conserved amino acid sequences surrounding predicted sulfated tyrosine sites. Tyrosine sulfation appears to be much more common in animal viruses than is currently recognized.