Shu-g Chen

CCR6 mediates dendritic cell localization, lymphocyte homeostasis, and immune responses in mucosal tissue.

Chemokine-directed migration of leukocyte subsets may contribute to the qualitative differences between systemic and mucosal immunity. Here, we demonstrate that in mice lacking the chemokine receptor CCR6, dendritic cells expressing CD11c and CD11b are absent from the subepithelial dome of Peyers patches. These mice also have an impaired humoral immune response to orally administered antigen and to the enteropathic virus rotavirus. In addition, CCR6(-/-) mice have a 2-fold to 15-fold increase in cells of select T lymphocyte populations within the mucosa, including CD4+ and CD8+ alphabeta-TCR T cells. By contrast, systemic immune responses to subcutaneous antigens in CCR6(-/-) mice are normal. These findings demonstrate that CCR6 is a mucosa-specific regulator of humoral immunity and lymphocyte homeostasis in the intestinal mucosa.

Ubiquitous Transgenic Expression of the IL-23 Subunit p19 Induces Multiorgan Inflammation, Runting, Infertility, and Premature Death

p19, a molecule structurally related to IL-6, G-CSF, and the p35 subunit of IL-12, is a subunit of the recently discovered cytokine IL-23. Here we show that expression of p19 in multiple tissues of transgenic mice induced a striking phenotype characterized by runting, systemic inflammation, infertility, and death before 3 mo of age. Founder animals had infiltrates of lymphocytes and macrophages in skin, lung, liver, pancreas, and the digestive tract and were anemic. The serum concentrations of the proinflammatory cytokines TNF-α and IL-1 were elevated, and the number of circulating neutrophils was increased. In addition, ubiquitous expression of p19 resulted in constitutive expression of acute phase proteins in the liver. Surprisingly, liver-specific expression of p19 failed to reproduce any of these abnormalities, suggesting specific requirements for production of biologically active p19. Bone marrow transfer experiments showed that expression of p19 by hemopoietic cells alone recapitulated the phenotype induced by its widespread expression, pointing to hemopoietic cells as the source of biologically active p19. These findings indicate that p19 shares biological properties with IL-6, IL-12, and G-CSF and that cell-specific expression is required for its biological activity.

Ectopic Expression of the Murine Chemokines CCL21a and CCL21b Induces the Formation of Lymph Node-Like Structures in Pancreas, But Not Skin, of Transgenic Mice

The CC chemokine CCL21 is a potent chemoattractant for lymphocytes and dendritic cells in vitro. In the murine genome there are multiple copies of CCL21 encoding two CCL21 proteins that differ from each other by one amino acid at position 65 (either a serine or leucine residue). In this report, we examine the expression pattern and biological activities of both forms of CCL21. We found that although both serine and leucine forms are expressed in most tissues examined, the former was the predominant form in lymphoid organs while the latter was predominantly expressed in nonlymphoid organs. When expressed in transgenic pancreas, both forms of CCL21 were capable of inducing the formation of lymph node-like structures composed primarily of T and B cells and a few dendritic cells. Induction of lymph node-like structures by these CCL21 proteins, however, could not be reproduced in every tissue. For instance, no lymphocyte recruitment or accumulation was observed when CCL21 was overexpressed in the skin. We conclude that both forms of CCL21 protein are biologically equivalent in promoting lymphocyte recruitment to the pancreas, and that their ability to induce the formation of lymph node-like structures is dependent on the tissues in which they are expressed.

Generation and analysis of mice lacking the chemokine fractalkine.

ABSTRACT Fractalkine (CX3CL1) is the first described chemokine that can exist either as a soluble protein or as a membrane-bound molecule. Both forms of fractalkine can mediate adhesion of cells expressing its receptor, CX3CR1. This activity, together with its expression on endothelial cells, suggests that fractalkine might mediate adhesion of leukocytes to the endothelium during inflammation. Fractalkine is also highly expressed in neurons, and its receptor, CX3CR1, is expressed on glial cells. To determine the biologic role of fractalkine, we used targeted gene disruption to generate fractalkine-deficient mice. These mice did not exhibit overt behavioral abnormalities, and histologic analysis of their brains did not reveal any gross changes compared to wild-type mice. In addition, these mice had normal hematologic profiles except for a decrease in the number of blood leukocytes expressing the cell surface marker F4/80. The cellular composition of their lymph nodes did not differ significantly from that of wild-type mice. Similarly, the responses offractalkine−/− mice to a variety of inflammatory stimuli were indistinguishable from those of wild-type mice.

Tumorigenesis induced by the HHV8-encoded chemokine receptor requires ligand modulation of high constitutive activity.

ORF74 (or KSHV-vGPCR) is a highly constitutively active G protein-coupled receptor encoded by HHV8 that is regulated both positively and negatively by endogenous chemokines. When expressed in transgenic mice, this chemokine receptor induces an angioproliferative disease closely resembling Kaposi sarcoma (KS). Here we demonstrate that several lines of mice carrying mutated receptors deficient in either constitutive activity or chemokine regulation fail to develop KS-like disease. In addition, animals expressing a receptor that preserves chemokine binding and constitutive activity but that does not respond to agonist stimulation have a much lower incidence of angiogenic lesions and tumors. These results indicate that induction of the KS-like disease in transgenic mice by ORF74 requires not only high constitutive signaling activity but also modulation of this activity by endogenous chemokines.

A Novel Model for Lymphocytic Infiltration of the Thyroid Gland Generated by Transgenic Expression of the CC Chemokine CCL21

Lymphocytic infiltrates and lymphoid follicles with germinal centers are often detected in autoimmune thyroid disease (AITD), but the mechanisms underlying lymphocyte entry and organization in the thyroid remain unknown. We tested the hypothesis that CCL21, a chemokine that regulates homeostatic lymphocyte trafficking, and whose expression has been detected in AITD, is involved in the migration of lymphocytes to the thyroid. We show that transgenic mice expressing CCL21 from the thyroglobulin promoter (TGCCL21 mice) have significant lymphocytic infiltrates, which are topologically segregated into B and T cell areas. Although high endothelial venules expressing peripheral lymph node addressin were frequently observed in the thyroid tissue, lymphocyte recruitment was independent of L-selectin or lymphotoxin-α but required CCR7 expression. Taken together, these results indicate that CCL21 is sufficient to drive lymphocyte recruitment to the thyroid, suggest that CCL21 is involved in AITD pathogenesis, and establish TGCCL21 transgenic mice as a novel model to study the formation and function of lymphoid follicles in the thyroid.

Inflammatory Signals shift from adipose to liver during high fat feeding and influence the development of steatohepatitis in mice

BackgroundObesity and inflammation are highly integrated processes in the pathogenesis of insulin resistance, diabetes, dyslipidemia, and non-alcoholic fatty liver disease. Molecular mechanisms underlying inflammatory events during high fat diet-induced obesity are poorly defined in mouse models of obesity. This work investigated gene activation signals integral to the temporal development of obesity.MethodsGene expression analysis in multiple organs from obese mice was done with Taqman Low Density Array (TLDA) using a panel of 92 genes representing cell markers, cytokines, chemokines, metabolic, and activation genes. Mice were monitored for systemic changes characteristic of the disease, including hyperinsulinemia, body weight, and liver enzymes. Liver steatosis and fibrosis as well as cellular infiltrates in liver and adipose tissues were analyzed by histology and immunohistochemistry.ResultsObese C57BL/6 mice were fed with high fat and cholesterol diet (HFC) for 6, 16 and 26 weeks. Here we report that the mRNA levels of macrophage and inflammation associated genes were strongly upregulated at different time points in adipose tissues (6-16 weeks) and liver (16-26 weeks), after the start of HFC feeding. CD11b+ and CD11c+ macrophages highly infiltrated HFC liver at 16 and 26 weeks. We found clear evidence that signals for IL-1β, IL1RN, TNF-α and TGFβ-1 are present in both adipose and liver tissues and that these are linked to the development of inflammation and insulin resistance in the HFC-fed mice.ConclusionsMacrophage infiltration accompanied by severe inflammation and metabolic changes occurred in both adipose and liver tissues with a temporal shift in these signals depending upon the duration of HFC feeding. The evidences of gene expression profile, elevated serum alanine aminotransferase, and histological data support a progression towards nonalcoholic fatty liver disease and steatohepatitis in these HFC-fed mice within the time frame of 26 weeks.

Disruption of Neutrophil Migration in a Conditional Transgenic Model: Evidence for CXCR2 Desensitization In Vivo

We developed transgenic mice conditionally expressing the neutrophil chemoattracting chemokine KC and the β-galactosidase gene in multiple tissues. In these transgenic mice, doxycycline treatment induced a strong up-regulation in the expression of KC in several tissues, including heart, liver, kidney, skin, and skeletal muscle. Expression of KC within these tissues led to a rapid and substantial increase in the serum levels of KC (serum KC levels were higher than 200 ng/ml 24 h after treatment). Accordingly, β-galactosidase expression was also detected after injection of doxycycline and was highest in skeletal muscle, pancreas, and liver. Surprisingly, despite expression of KC in multiple tissues, no neutrophil infiltration was observed in any of the tissues examined, including skin. Doxycycline treatment of nontransgenic mice grafted with transgenic skin caused dense neutrophilic infiltration of the grafts, but not the surrounding host skin, indicating that the KC produced in transgenic tissues was biologically active. In separate experiments, neutrophil migration toward a localized source of recombinant KC was impaired in animals overexpressing KC but was normal in response to other neutrophil chemoattractants. Analysis of transgenic neutrophils revealed that high concentrations of KC in transgenic blood had no influence on L-selectin cell surface expression but caused desensitization of the receptor for KC, CXCR2. These results confirm the neutrophil chemoattractant properties of KC and provide a mechanistic explanation for the paradoxical lack of leukocyte infiltration observed in the presence of elevated concentrations of this chemokine.

Central Nervous System Inflammation and Neurological Disease in Transgenic Mice Expressing the CC Chemokine CCL21 in Oligodendrocytes

To study the biological role of the chemokine ligands CCL19 and CCL21, we generated transgenic mice expressing either gene in oligodendrocytes of the CNS. While all transgenic mice expressing CCL19 in the CNS developed normally, most (18 of 26) of the CCL21 founder mice developed a neurological disease that was characterized by loss of landing reflex, tremor, and ataxia. These neurological signs were observed as early as postnatal day 9 and were associated with weight loss and death during the first 4 wk of life. Microscopic examination of the brain and spinal cord of CCL21 transgenic mice revealed scattered leukocytic infiltrates that consisted primarily of neutrophils and eosinophils. Additional findings included hypomyelination, spongiform myelinopathy with evidence of myelin breakdown, and reactive gliosis. Thus, ectopic expression of the CC chemokine CCL21, but not CCL19, induced a significant inflammatory response in the CNS. However, neither chemokine was sufficient to recruit lymphocytes into the CNS. These observations are in striking contrast to the reported activities of these molecules in vitro and may indicate specific requirements for their biological activity in vivo.

Impaired Pulmonary Host Defense in Mice Lacking Expression of the CXC Chemokine Lungkine

Lungkine (CXCL15) is a novel CXC chemokine that is highly expressed in the adult mouse lung. To determine the biologic function of Lungkine, we generated Lungkine null mice by targeted gene disruption. These mice did not differ from wild-type mice in their hematocrits or in the relative number of cells in leukocyte populations of peripheral blood or other tissues, including lung and bone marrow. However, Lungkine null mice were more susceptible to Klebsiella pneumonia infection, with a decreased survival and increased lung bacterial burden compared with infected wild-type mice. Histologic analysis of the lung and assessment of leukocytes in the bronchioalveolar lavage revealed that neutrophil numbers were normal in the lung parenchyma, but reduced in the airspace. The production of other neutrophil chemoattractants in the Lungkine null mice did not differ from that in wild-type mice, and neutrophil migration into other tissues was normal. Taken together, these findings demonstrate that Lungkine is an important mediator of neutrophil migration from the lung parenchyma into the airspace.