Developmental switches in chemokine response profiles during B cell differentiation and maturation.

Developing B cells undergo dramatic changes in their responses to chemoattractant cytokines (chemokines) and in expression of chemokine receptors. Bone marrow pre-pro-B cells (AA4.1(+)/natural killer 1.1(-) Fraction A cells) and cells capable of generating pro-B colonies in the presence of interleukin 7 and flt3 ligand migrate to thymus-expressed chemokine (TECK), a response lost in later stages of B cell development. B cell-attracting chemokine 1 (BCA-1) responses correlate with CXC chemokine receptor (CXCR)5 expression, are first displayed by a pro-B cell subset, are lost in pre-B cells, and then are regained just before and after egress from the marrow. All peripheral B cell subsets, including follicular and germinal center as well as marginal zone and peritoneal B1 B cells, respond to BCA-1, implying that responsiveness to this follicular chemokine is not sufficient to predict follicle localization. Responses to the CC chemokine receptor (CCR)7 ligands secondary lymphoid tissue chemoattractant (SLC) and macrophage inflammatory protein (MIP)-3beta, implicated in homing to lymphoid tissues, are upregulated before B cell exit from the marrow, but increase further in the periphery and are shared by all peripheral B cells. In contrast, responsiveness to MIP-3alpha and expression of CCR6 are acquired only after emigration to the periphery and during maturation into the recirculating B cell pool. Chemotaxis to stromal cell-derived factor 1alpha is observed at all stages of B cell differentiation. Thus, unique patterns of chemokine responses may help define developing B cell populations and direct their maturation in the marrow and migration to the periphery.

Monoclonal antibodies specific for beta 7 integrin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) reduce inflammation in the colon of scid mice reconstituted with CD45RBhigh CD4+ T cells.

Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is an adhesion protein expressed on endothelium in mucosal tissues that has been shown to play an important role in the selective homing of lymphocytes to intestinal mucosa and associated lymphoid tissue. To determine whether MAdCAM-1 or its ligand alpha 4 beta 7 would be appropriate targets for therapeutic intervention in gut-associated inflammation, we tested the ability of rat mAbs specific for beta 7 integrin and MAdCAM-1 to inhibit chronic colonic inflammation in scid mice reconstituted with CD4+ T cells enriched for the CD45RBhigh subpopulation. Abs specific for beta 7 and MAdCAM-1 blocked recruitment of lymphocytes to the colitic colon, and more importantly, these Abs significantly reduced the severity of colonic inflammatory disease in this animal model. Therefore, the adhesive interactions mediated by alpha 4 beta 7 and MAdCAM are intimately involved in leukocyte recruitment to gut in chronic inflammatory disease and may be relevant therapeutic targets for patients with inflammatory bowel disease.

Local expression of transgene encoded TNF alpha in islets prevents autoimmune diabetes in nonobese diabetic (NOD) mice by preventing the development of auto-reactive islet-specific T cells.

Lately, TNF alpha has been the focus of studies of autoimmunity; its role in the progression of autoimmune diabetes is, however, still unclear. To analyze the effects of TNF alpha in insulin-dependent diabetes mellitus (IDDM), we have generated nonobese diabetic (NOD) transgenic mice expressing TNF alpha under the control of the rat insulin II promoter (RIP). In transgenic mice, TNF alpha expression on the islets resulted in massive insulitis, composed of CD4+ T cells, CD8+ T cells, and B cells. Despite infiltration of considerable number of lymphoid cells in islets, expression of TNF alpha protected NOD mice from IDDM. To determine the mechanism of TNF alpha action, splenic cells from control NOD and RIP-TNF alpha mice were adoptively transferred to NOD-SCID recipients. In contrast to the induction of diabetes by splenic cells from control NOD mice, splenic cells from RIP-TNF alpha transgenic mice did not induce diabetes in NOD-SCID recipients. Diabetes was induced however, in the RIP-TNF alpha transgenic mice when CD8+ diabetogenic cloned T cells or splenic cells from diabetic NOD mice were adoptively transferred to these mice. Furthermore, expression of TNF alpha in islets also downregulated splenic cell responses to autoantigens. These data establish a mechanism of TNF alpha action and provide evidence that local expression of TNF alpha protects NOD mice from autoimmune diabetes by preventing the development of autoreactive islet-specific T cells.

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.

Local production of human IL-6 promotes insulitis but retards the onset of insulin-dependent diabetes mellitus in non-obese diabetic mice.

We produced transgenic mice which overexpress human IL-6 in pancreatic beta cells of C57BL/6 x CBA and non-obese diabetic (NOD) mice. Transgenic C57BL/6 x CBA mice back-crossed onto a C57BL/6 background do not develop insulitis or diabetes. In contrast, NOD/F1 transgenic mice develop a lymphocytic infiltrate of pancreatic islets which is not seen in negative littermates. Immunohistochemistry reveals these cells to be predominantly CD4+, CD8+, B220+ cells. Despite the insulitis, these mice do not develop diabetes. Transgenic rat insulin promoter-IL-6 mice were therefore also made on an inbred NOD background. These mice showed no difference in the onset or extent of insulitis when compared with non-transgenic NOD mice and no difference was found in the phenotype of the infiltrating cells. However, transgenic NOD mice had lower average fasting glucose levels and delayed onset of diabetes compared with age and sex matched littermate negative NOD mice. As a consequence, transgenic NOD mice also had longer survival than littermate negative NOD mice. We conclude that the expression of IL-6 by beta cells does not cause insulitis or diabetes in C57BL/6 x CBA mice, but that the interaction of IL-6 and diabetes susceptibility genes causes insulitis in NOD/F1 mice. Since IL-6 delays the onset of diabetes and prolongs survival of NOD mice, it is possible that the protective effect is caused by local IL-6 action on the islets, by the infiltrating lymphocytes or both.

Airway epithelial cell expression of interleukin-6 in transgenic mice. Uncoupling of airway inflammation and bronchial hyperreactivity.

We produced transgenic mice which overexpress human IL-6 in the airway epithelial cells. Transgenic mice develop a mononuclear cell infiltrate adjacent to large and mid-sized airways. Immunohistochemistry reveals these cells to be predominantly CD4+ cells, MHC class II+ cells, and B220+ cells. Transgenic mice and nontransgenic mice had similar baseline respiratory system resistance (0.47 +/- 0.06 vs 0.43 +/- 0.04 cmH2O/ml per s at 9 wk of age, P = NS and 0.45 +/- 0.07 vs 0.43 +/- 0.09 cmH2O/ml per s at 17 wk of age, P = NS). Transgenic mice, however, required a significantly higher log dose of methacholine to produce a 100% increase in respiratory system resistance as compared with non-transgenic littermates (1.34 +/- 0.24 vs 0.34 +/- 0.05 mg/ml, P < or = 0.01). We conclude that the expression of human IL-6 in the airways of transgenic mice results in a CD4+, MHC class II+, B220+ lymphocytic infiltrate surrounding large and mid-sized airways that does not alter basal respiratory resistance, but does diminish airway reactivity to methacholine. These findings demonstrate an uncoupling of IL-6-induced airway lymphocytic inflammation and airway hyperresponsiveness and suggest that some forms of airway inflammation may serve to restore altered airway physiology.

Costimulator B7-1 confers antigen-presenting-cell function to parenchymal tissue and in conjunction with tumor necrosis factor alpha leads to autoimmunity in transgenic mice.

Tolerance to peripheral antigens is thought to result from the inability of parenchymal tissue to stimulate T cells--an inability that is believed to relate to the lack of expression of the costimulatory signal(s) required for T-cell activation. To test this model, we generated transgenic mice expressing costimulatory molecule B7-1 on the B cells of the pancreas. We find that islets from these transgenic mice are immunogenic for naive T cells in vitro and in vivo. Nonetheless, mice expressing the costimulator B7-1 specifically on beta cells do not develop diabetes, suggesting that expression of the B7-1 costimulator is not sufficient to abrogate the tolerance to peripheral antigens. We have reported that tumor necrosis factor alpha subunit (TNF-alpha) expressed by beta cells leads to a local inflammation but no islet destruction. Strikingly, however, the combination of a local inflammation due to the expression of the cytokine TNF-alpha and the expression of B7-1 results in tissue destruction and diabetes.

Transgenic tumor necrosis factor (TNF)-alpha production in pancreatic islets leads to insulitis, not diabetes. Distinct patterns of inflammation in TNF-alpha and TNF-beta transgenic mice.

To understand the role of TNF in the regulation of inflammation and the development of autoimmune diseases such as insulin-dependent diabetes mellitus, we produced transgenic mice in which the synthesis of murine TNF-alpha was directed by the rat insulin II promoter. The expression of the TNF-alpha transgene was restricted to the pancreas, in contrast to TNF-beta expression from the same promoter, in which the transgene was expressed in the pancreas, kidney, and skin. The expression of TNF-alpha in the pancreas of transgenic mice resulted in an overwhelming insulitis, composed of CD4+ and CD8+ T cells and B220+ B cells, considerably greater than that of TNF-beta transgenics. Moreover, in contrast to the predominant peri-insulitis observed in TNF-beta transgenic mice, the majority of the infiltrate in the TNF-alpha transgenic mice was within the islet itself. These unique patterns of infiltration were observed in the F1 progeny of crosses with C57BL/6 as well as NOD. Both TNF-alpha and TNF-beta transgenic mice show elevated expression of leukocyte adhesion molecules VCAM-1 and ICAM-1 in islet endothelia and increased expression of MHC class I on islet cells. This inflammation did not result in reduced insulin content of the islets, nor did it lead to diabetes. These data suggest that additional stimuli are necessary to initiate the process of islet destruction.

Insulitis in transgenic mice expressing tumor necrosis factor beta (lymphotoxin) in the pancreas.

Tumor necrosis factor beta (TNF-beta) (lymphotoxin) may play an important role in the immune response and pathologic inflammatory diseases. Insulitis is an important early step in the development of insulin-dependent diabetes mellitus. To understand better the role of TNF-beta in the regulation of inflammation and type 1 diabetes, we produced transgenic mice in which the murine TNF-beta gene was regulated by the rat insulin II promoter. The transgene was expressed in the pancreas, kidney, and skin of transgenic mice. The expression of TNF-beta in the pancreas of transgenic mice resulted in a leukocytic inflammatory infiltrate consisting primarily of B220+ IgM+ B cells and CD4+ and CD8+ T cells. The insulitis is reminiscent of the early stages of diabetes, though the mice did not progress to diabetes.

Allelic exclusion of membrane but not secreted immunoglobulin in a mature B cell line.

Although many B lymphocytes contain two completely rearranged immunoglobulin (Ig) heavy (H) chain genes, only one of the alleles specifies a protein product. This phenomenon is termed allelic exclusion and is controlled in part by the membrane portion of Ig H chains. We have identified a mature B cell line derived from murine bone marrow that expresses two H chain proteins, gamma 3 and gamma 2b. Proteins of appropriate size for the membrane and secreted forms of both H chains are produced and both IgG3 and IgG2b are secreted from the cells. However, only IgG3 is expressed on the membrane. Detailed restriction mapping of the H chain genes indicates that both are rearranged, one containing a VHJ558 gene linked to the gamma 3 constant region and the other a VHS107 gene linked to the gamma 2b constant region. Primer extension sequencing of the RNA reveals that the gamma 2b RNA is transcribed from the allele containing VHS107 sequences while the gamma 3 RNA is transcribed from the allele containing VHJ558 sequences. Thus, this mature B cell line secretes two distinct antibody molecules but is allelically excluded at the level of surface Ig expression. This cell line provides a model system to study factors, in addition to aberrant rearrangement, that influence allelic exclusion.