ICOS co-stimulatory receptor is essential for T-cell activation and function.

T-lymphocyte activation and immune function are regulated by co-stimulatory molecules. CD28, a receptor for B7 gene products, has a chief role in initiating T-cell immune responses. CTLA4, which binds B7 with a higher affinity, is induced after T-cell activation and is involved in downregulating T-cell responses. The inducible co-stimulatory molecule (ICOS), a third member of the CD28/CTLA4 family, is expressed on activated T cells. Its ligand B7H/B7RP-1 is expressed on B cells and in non-immune tissues after injection of lipopolysaccharide into animals. To understand the role of ICOS in T-cell activation and function, we generated and analysed ICOS-deficient mice. Here we show that T-cell activation and proliferation are defective in the absence of ICOS. In addition, ICOS -/- T cells fail to produce interleukin-4 when differentiated in vitro or when primed in vivo. ICOS is required for humoral immune responses after immunization with several antigens. ICOS-/- mice showed greatly enhanced susceptibility to experimental autoimmune encephalomyelitis, indicating that ICOS has a protective role in inflammatory autoimmune diseases.

Conditional vascular cell adhesion molecule 1 deletion in mice: impaired lymphocyte migration to bone marrow.

We generated vascular cell adhesion molecule (VCAM)-1 "knock-in" mice and Cre recombinase transgenic mice to delete the VCAM-1 gene (vcam-1) in whole mice, thereby overcoming the embryonic lethality seen with conventional vcam-1-deficient mice. vcam-1 knock-in mice expressed normal levels of VCAM-1 but showed loss of VCAM-1 on endothelial and hematopoietic cells when interbred with a "TIE2Cre" transgene. Analysis of peripheral blood from conditional vcam-1-deficient mice revealed mild leukocytosis, including elevated immature B cell numbers. Conversely, the bone marrow (BM) had reduced immature B cell numbers, but normal numbers of pro-B cells. vcam-1-deficient mice also had reduced mature IgD+ B and T cells in BM and a greatly reduced capacity to support short-term migration of transferred B cells, CD4+ T cells, CD8+ T cells, and preactivated CD4+ T cells to the BM. Thus, we report an until now unappreciated dominant role for VCAM-1 in lymphocyte homing to BM.

Cutting edge: critical role of inducible costimulator in germinal center reactions.

Inducible costimulator (ICOS) is a new member of the CD28/CTLA-4 family that is expressed on activated and germinal center (GC) T cells. Recently, we reported that ICOS-deficient mice exhibited profound defects in T cell activation and effector function. Ab responses in a T-dependent primary reaction and in a murine asthma model were also diminished. In the current study, we investigate the mechanism by which ICOS regulates humoral immunity and examine B cell GC reactions in the absence of ICOS. We found that ICOS(-/-) mice, when immunized with SRBC, had smaller GCs. Furthermore, IgG1 class switching in the GCs was impaired. Remarkably, GC formation in response to a secondary recall challenge was completely absent in ICOS knockout mice. These data establish a critical role of ICOS in regulation of humoral immunity.

Expression of interleukin 9 in the lungs of transgenic mice causes airway inflammation, mast cell hyperplasia, and bronchial hyperresponsiveness.

Interleukin (IL)-9, a pleiotropic cytokine produced by the Th2 subset of T lymphocytes has been proposed as product of a candidate gene responsible for asthma. Its wide range of biological functions on many cell types involved in the allergic immune response suggests a potentially important role in the complex pathogenesis of asthma. To investigate the contributions of IL-9 to airway inflammation and airway hyperresponsiveness in vivo, we created transgenic mice in which expression of the murine IL-9 cDNA was regulated by the rat Clara cell 10 protein promoter. Lung selective expression of IL-9 caused massive airway inflammation with eosinophils and lymphocytes as predominant infiltrating cell types. A striking finding was the presence of increased numbers of mast cells within the airway epithelium of IL-9-expressing mice. Other impressive pathologic changes in the airways were epithelial cell hypertrophy associated with accumulation of mucus-like material within nonciliated cells and increased subepithelial deposition of collagen. Physiologic evaluation of IL-9-expressing mice demonstrated normal baseline airway resistance and markedly increased airway hyperresponsiveness to inhaled methacholine. These findings strongly support an important role for IL-9 in the pathogenesis of asthma.

Interleukin-4 alters epithelial cell differentiation and surfactant homeostasis in the postnatal mouse lung.

Interleukin-4 (IL-4) is a pleotrophic cytokine which is increased during lung injury and inflammation. Epithelial cell morphology and surfactant homeostasis were assessed in 4-52-wk-old transgenic mice in which IL-4 was expressed in the bronchial and bronchiolar epithelial cells under the control of the Clara cell secretory protein promoter (CCSP-IL-4 mice). IL-4 caused progressive pulmonary infiltration with macrophages, lymphocytes, neutrophils, and eosinophils. Epithelial cell hypertrophy and mucus cell metaplasia were observed in the lungs of CCSP-IL-4 mice at all ages. Airway epithelial cells contained increased neutral glycoproteins and expressed gastric mucin, normally absent in the bronchiolar epithelium of the mouse. Immunohistochemical and biochemical studies demonstrated increased surfactant proteins A and B in lung sections and lung homogenates of CCSP-IL-4 transgenic mice. Increased immunostaining for surfactant proprotein C was also detected in type II epithelial cells of the transgenic mice. In contrast, surfactant protein B and CCSP expression was decreased or was absent in hypertrophic epithelial cells lining the conducting airways of transgenic mice. Lung-specific increase in T-cell proliferative responses to mitogenic stimulation and antibody secretion were detected in CCSP-IL-4 mice. Differentiated characteristics of respiratory epithelial cells were dramatically influenced by the chronic production of IL-4 in the conducting airways. Alterations in lung morphology in the CCSP-IL-4 mice are similar to some of those induced by antigenic stimulation or associated with chronic airway inflammation.

A novel role for murine IL-4 in vivo: induction of MUC5AC gene expression and mucin hypersecretion.

Mucus hypersecretion and plugging of lower respiratory tract airways contributes to the morbidity and mortality associated with asthma. Interleukin (IL)-4 plays a putative role in some forms of asthma. Thus, transgenic mice that overexpress murine IL-4 selectively within the lung were used to study the effect of IL-4 on mucus glycoprotein gene expression and mucin release. Histologic examination of lung sections from IL-4 mice revealed that nonciliated epithelial cells from conducting airways were hypertrophic, due at least in part to the accumulation of mucus glycoprotein. The cytoplasm of these cells stained positively for glycoproteins using mucicarmine, alcian blue (AB), and periodic acid-Schiff (PAS). Ciliated cells were also enlarged but did not show any mucin-specific staining. Inclusion granules typically found in nonciliated (Clara) cells of control mice were absent in the IL-4 transgenic mice. Northern blot analysis of total RNA from lung tissue revealed that the expression of the MUC5AC, but not MUC2, mucin gene was distinctly upgraded in IL-4 transgenic mice compared to transgene-negative controls. In addition, a 5- to 10-fold increase in AB- and PAS-positive material was found in lavage fluid from IL-4 overexpressing mice compared to transgene-negative controls. Thus, the overexpression of IL-4 locally within the lung enhances mucus glycoprotein synthesis by altering gene expression, results in the accumulation of mucus glycoprotein in nonciliated epithelial cells, and induces the release of mucus into the airway lumen. We therefore hypothesize that the overproduction of mucus seen in some patients with asthma may be a direct result of the action of IL-4 within the inflamed lung.

Phenotypic and physiologic characterization of transgenic mice expressing interleukin 4 in the lung: lymphocytic and eosinophilic inflammation without airway hyperreactivity.

To investigate the contribution of interleukin-4 (IL-4) to airway inflammation in vivo and to explore directly its relationship to airway reactivity, we created transgenic mice in which the murine cDNA for IL-4 was regulated by the rat Clara cell 10 protein promoter. Expression was detected only in the lung and not in thymus, heart, liver, spleen, kidney, or uterus. The expression of IL-4 elicited hypertrophy of epithelial cells of the trachea, bronchi, and bronchioles. Hypertrophy is due, at least in part, to the accumulation of mucus glycoprotein. Histologic examination of parenchyma revealed multinucleated macrophages and occasional islands of cells consisting largely of eosinophils or lymphocytes. Analysis of lung lavage fluid revealed the presence of a leukocytic infiltrate consisting of lymphocytes, neutrophils and eosinophils. Mice expressing IL-4 had greater baseline airway resistance but did not demonstrate hyperreactivity to methacholine. Thus, the expression of IL-4 selectively within the lung elicits an inflammatory response characterized by epithelial cell hypertrophy, and the accumulation of macrophages, lymphocytes, eosinophils, and neutrophils without resulting in an alteration in airway reactivity to inhaled methacholine.