The absence of cutaneous lymph nodes results in a Th2 response and increased susceptibility to Leishmania major infection in mice.

Lymph nodes (LNs) are important sentinel organs where antigen-presenting cells interact with T cells to induce adaptive immune responses. In cutaneous infection of mice with Leishmania major, resistance depends on the induction of a T-helper-cell-1 (Th1)-mediated cellular immune response in draining, peripheral LNs. We investigated whether draining, peripheral LNs are absolutely required for resistance against L. major infection. We investigated the course of experimental leishmaniasis in wild-type (wt) mice lacking peripheral LNs (pLNs), which we generated by in utero blockade of membrane-bound lymphotoxin, and in mice lacking pLNs or all LNs due to genetic deletion of lymphotoxin ligands or receptors. wt mice of the resistant C57BL/6 strain without local skin-draining LNs were still able to generate specific T-cell responses, but this yielded Th2 cells. This switch to a Th2 response resulted in severe systemic infection. We also confirmed these results with mice lacking pLNs due to genetic depletion of lymphotoxin-beta. The complete absence of LNs due to a genetic depletion of the lymphotoxin-beta receptor also resulted in a marked deterioration of disease and a Th2 response. Thus, in the absence of pLNs, an L. major-specific Th2 response is induced in the remaining secondary lymphoid organs, such as the spleen and non-skin-draining LNs. This indicates a critical requirement for pLNs to induce protective Th1 immunity and suggests that whether Th1 or Th2 priming to the same antigen occurs depends on the site of the primary antigen recognition.

Role of lymphotoxins in the development of Peyer's patches and mesenteric lymph nodes: relevance to intestinal inflammation and treatment.

Hallmarks of the adaptive immune system are antigen-specific cellular and humoral immune responses. Secondary lymphoid organs serve as sites of contact between antigen-presenting cells (APCs) and immune effector T and B lymphocytes. The gut-associated lymphatic system (GALT) as the intestinal branch of the immune system provides different mechanisms to protect organisms against pathogens. Simultaneously, immune activation secondary to genetic factors and/or environmental signals can induce detrimental autoimmunity. The effector pathways in host defense and autoimmunity use similar cytokines and chemokines. Unlike few other cytokines, lymphotoxin (LT) alpha/beta regulates the development of intestinal lymphoid organs, including Peyer's patches (PPs) and mesenteric lymph nodes (MLNs). In addition, intestinal inflammation is suppressed by inhibition of LTbeta signalling, an observation which has initiated clinical studies using this treatment principle. Conversely, the course of Citrobacter rodentium-induced infectious colitis is more severe in mice with impaired LTbeta-receptor-mediated signalling. This report provides an overview on the role of the different organs of the GALT in intestinal inflammation. Moreover, it describes the role of the LTbeta-receptor-mediated signalling in intestinal inflammation as encountered in autoimmune and infectious pathology. The contribution of LT to the delicate balance of immune effector functions in host defense and autoimmunity is discussed.

Expression of vascular cell adhesion molecule-1 (CD 106) in normal and neoplastic human esophageal squamous epithelium.

Vascular cell adhesion molecule-1 (VCAM-1), a key receptor for the leukocyte-associated integrin (VLA4), is a crucial mediator of leukocyte adhesion and has co-stimulatory functions in inflammation at various organ sites. Specifically, VCAM-1/VLA4 interactions have been shown to play important roles in the setting of cutaneous immune responses, such as psoriatic lesions in humans and acute Graft-versus-Host-Disease in mice. VCAM-1 is generally expressed on activated endothelial cells in inflamed tissues, mediating endothelium-leukocyte interactions, leading to leukocyte diapedesis to the site of inflammation. We report novel and unexpected membrane expression of VCAM-1 in the basal squamous epithelial strata of the normal human esophagus and distinct patterns of epithelial expression in esophageal pathology. To further delineate the differential expression patterns of VCAM-1 in the esophageal epithelium, we examined specimens from squamous cell carcinoma (SCC), adenocarcinoma, and Barrett's columnar cell metaplasia. VCAM-1 was strongly expressed in squamous cell carcinoma, but not adenocarcinoma nor columnar epithelia in Barrett's esophagus. VCAM-1 expression was focally accentuated at sites characteristic of microscopic tumor invasion in SCC, pointing to a potential role of VCAM-1 in the development of metastasis. In addition, in vitro immunofluorescence studies using OE21 cells, an esophageal squamous epithelial cell line, displayed distinct VCAM-1 immunoreactivity confined to mitotic and dividing cells. Cell cycle arrest caused a significant decrease in VCAM-1 immunoreactivity in OE21 cells. These data suggest a previously unappreciated role for VCAM-1 in esophageal squamous epithelial homeostasis and pathology.

Absence of CCR6 inhibits CD4+ regulatory T-cell development and M-cell formation inside Peyer's patches.

The chemokine Mip3alpha is specifically expressed by the follicle-associated epithelia (FAE) covering intestinal Peyer's patches (PPs) and is the only known chemokine ligand for the chemokine receptor CCR6. Although CCR6-deficient mice are known to have a perturbed intestinal immune system, little is known about the specific impact of this interaction for Peyer's patch formation. To elucidate the effect of Mip3alpha on PP lymphocyte development, we used a CCR6/enhanced green fluorescent protein (EGFP) knock-in mouse model and analyzed lymphocyte development by immunohistochemistry and flow cytometry. PPs of CCR6-/- mice were significantly size-reduced with a proportional loss of B cells and T cells, whereas T-cell subsets were disturbed with a decreased CD4/CD8 ratio paralleled with a loss of regulatory CD4+ CD45Rb(low) T cells. The analysis of cytokine production by CCR6-expressing cells could demonstrate that CCR6 is involved in the regulation of cytokine secretion such as interleukin-12 by dendritic cells. Quantification of UEA-1+ cells inside the FAE showed reduced M-cell numbers in CCR6-deficient mice. These results suggest that the interaction of CCR6 with its ligand Mip3alpha is important for immune responses generated inside the PPs, particularly for the generation of regulatory CD4+ T cells residing inside PPs and for the formation of M cells.

The lymphotoxin-beta receptor is critical for control of murine Citrobacter rodentium-induced colitis.

BACKGROUND AND AIMS: Lymphotoxin is a tumor necrosis factor-family cytokine. Blocking of lymphotoxin alpha 1 beta 2 /lymphotoxin-beta receptor interactions prevents experimental colitis in mice, and this suggests a potential treatment principle of human inflammatory bowel disease. Infection of mice with Citrobacter rodentium serves as an animal model for human infectious colitis induced by enteropathogenic Escherichia coli . We studied the role of lymphotoxin alpha 1 beta 2 /lymphotoxin-beta receptor signaling in Citrobacter rodentium -induced colitis. METHODS: Mice with disrupted lymphotoxin alpha 1 beta 2 /lymphotoxin-beta receptor interactions secondary to gene defects (lymphotoxin-alpha -/- , lymphotoxin-beta -/- , and lymphotoxin-beta receptor -/- ) or treatment with the antagonist lymphotoxin-beta receptor-immunoglobulin G fusion protein were infected with Citrobacter rodentium . Body weight, fecal excretion of Citrobacter rodentium , and disease-related mortality were monitored. Spleen and liver organ cultures of mice assessed systemic infection. Intestinal inflammation and lymphoid architecture were histologically recorded in the large intestine, mesenteric lymph nodes, and spleen of infected mice. RESULTS: Inhibition of lymphotoxin alpha 1 beta 2 /lymphotoxin-beta receptor interactions was associated with increased severity of Citrobacter rodentium -induced colitis, as indicated by increased disease-related mortality, more severe weight loss, intestinal bacterial abscesses, and a higher burden of Citrobacter rodentium in the spleen and liver of -/- and lymphotoxin-beta receptor-immunoglobulin G-treated mice. There was a reduction of CD11c + dendritic cells in the spleen of naive and infected -/- and lymphotoxin-beta receptor-immunoglobulin G-treated mice. In infected lymphotoxin-beta receptor -/- mice, anti- Citrobacter rodentium immunoglobulin G2a levels were decreased, whereas immunoglobulin G1 levels were increased. Citrobacter rodentium -induced interleukin-4 secretion was increased in lymphotoxin-beta receptor -/- mice. CONCLUSIONS: Lymphotoxin alpha 1 beta 2 /lymphotoxin-beta receptor interactions are critical for immunity against Citrobacter rodentium in mice. Impaired anti-enteropathogenic Escherichia coli immunity may be anticipated in anti-lymphotoxin-beta receptor-directed therapy for human inflammatory bowel disease.

Human intestinal microvascular endothelial cells express Toll-like receptor 5: a binding partner for bacterial flagellin.

Bacterial flagellin has recently been identified as a ligand for Toll-like receptor 5 (TLR5). Human sites known to specifically express TLR5 include macrophages and gastric and intestinal epithelium. Because infection of intestinal epithelial cells with Salmonella leads to an active transport of flagellin to the subepithelial compartment in proximity to microvessels, we hypothesized that human intestinal endothelial cells functionally express TLR5, thus enabling an active inflammatory response upon binding of translocated flagellin. Endothelial expression of TLR5 in human macro- and microvascular endothelial cells was examined by RT-PCR, immunoblot analysis, and immunofluorescence. Endothelial expression of TLR5 in vivo was verified by immunohistochemistry. Endothelial modulation of ICAM-1 expression was quantitated using flow cytometry, and leukocyte transmigration in vitro was assessed by an endothelial transmigration assay. Epithelial-endothelial cellular interactions upon infection with viable Salmonella were investigated using a coculture system in vitro. We found that Salmonella-infected intestinal epithelial cells induce endothelial ICAM-1 expression in cocultured human endothelial cells. Both macro- (HUVEC) and microvascular endothelial cells derived from human skin (human dermal microvascular endothelial cell 1) and human colon (human intestinal microvascular endothelial cells) were found to express high constitutive amounts of TLR5 mRNA and protein. These findings were paralleled by strong immunoreactivity for TLR5 of normal human colonic microvessels in vivo. Furthermore, incubation of human dermal microvascular endothelial cells with flagellin from clinical isolates of Escherichia and Salmonella strains led to a marked up-regulation of ICAM-1, as well as to an enhanced leukocyte transendothelial cell migration. These results suggest that endothelially expressed TLR5 might play a previously unrecognized role in the innate immune response toward bacterial Ags.

Induction of colitis in mice deficient of Peyer's patches and mesenteric lymph nodes is associated with increased disease severity and formation of colonic lymphoid patches.

Inflammatory bowel disease is associated with immune activation in Peyer's patches and mucosal lymph nodes. The role of these organs in dextran sodium sulfate (DSS)-induced colitis was investigated. We used mice lacking Peyer's patches and/or lymph nodes because of lymphotoxin-alpha gene deficiency or treatment in utero with lymphotoxin-beta-receptor IgG and tumor necrosis factor-receptor-I (55)-IgG fusion proteins. Mice lacking Peyer's patches and lymph nodes because of lymphotoxin-alpha deficiency or in utero fusion protein treatment developed more severe colitis than control mice as indicated by more severe intestinal shrinking, longer colonic ulcers, and higher histological disease scores. Oral DSS triggered the formation of colonic submucosal lymphoid patches in these mice and caused an increase in the number of submucosal lymphoid patches in mice treated in utero with the fusion proteins. Mice lacking Peyer's patches only showed more submucosal lymphoid patches whereas intestinal length and histological disease score were similar to control mice. In conclusion, more severe DSS-induced colitis correlates with the loss of the mesenteric lymph nodes. However, neither the absence of Peyer's patches nor the presence of colonic lymphoid patches were correlated with increased disease severity.

Mesenteric lymph nodes are critical for the induction of high-dose oral tolerance in the absence of Peyer's patches.

We have previously demonstrated the loss of oral tolerance (OT) in lymphotoxinalpha-/- (LTalpha-/-) and TNFalpha / lymphotoxinalpha deficient (TNFalpha / LTalpha-/-) mice which have defective Peyer's patches (PP) and lymph node (LN) development. We have now studied OT in BALB / c mice with differential defects of the gut-associated lymphoid tissue (GALT) caused by inhibition of LTbetaR signaling during fetal development. Treatment of pregnant mice with LTbetaR-IgG (LTbetaRIgG) and TNFR I55-IgG (TNFR55IgG) abrogates the formation of PP (LTbetaRIgG) or of PP and mesenteric LN (MLN) (LTbetaRIgG / TNFRIgG) without genetically deleting the respective cytokine pathways. OT was readily induced in mice without PP but retaining MLN (PP null / LN +). In contrast, OTcould not be induced in mice lacking both MLN and PP (PP null / MLN null) as shown by the inability of these mice to suppress IFN-gamma secretion or DTH reactions. We next assessed OT in 129 x B6 LTalpha-/- mice with and without MLN. Timed treatment of pregnant LTalpha-/- mice with an agonist anti-LTbetaR mAb induces formation of MLN but not of PP in LTalpha-/- mice. LN + LTalpha-/- mice developed OT while LN LTalpha-/- mice were resistant to OT induction. Taken collectively, the data show that in the presence of MLN PP are not required for OT induction and that the presence of MLN is sufficient for OT induction in the LTalpha-/- model.