Differential Regulation of Mouse B Cell Development by Transforming Growth Factor β1

Transforming growth factor β (TGFβ) can inhibit thein vitroproliferation, survival and differentiation of B cell progenitors, mature B lymphocytes and plasma cells. Here we demonstrate unexpected, age-dependent reductions in the bone marrow (BM) B cell progenitors and immature B cells in TGFβ1-/-mice. To evaluate TGFβ responsiveness during normal B lineage development, cells were cultured in interleukin 7 (IL7)±TGFβ. Picomolar doses of TGFβ1 reduced pro-B cell recoveries at every timepoint. By contrast, the pre-B cells were initially reduced in number, but subsequently increased compared to IL7 alone, resulting in a 4-fold increase in the growth rate for the pre-B cell population. Analysis of purified BM sub-populations indicated that pro-B cells and the earliest BP1-pre-B cells were sensitive to the inhibitory effects of TGFβ1. However, the large BP1+pre-B cells, although initially reduced, were increased in number at days 5 and 7 of culture. These results indicate that TGFβ1 is important for normal B cell developmentin vivo, and that B cell progenitors are differentially affected by the cytokine according to their stage of differentiation.

Different Roles of a Rat Cortical Thymic Epithelial Cell LineIn Vitroon Thymocytes and Thymocyte Hybridoma Cells: Phagocytosis, Induction of Apoptosis, Nursing and Growth Promoting Activities

In this work, the interaction between a rat cortical thymic epithelial cell (TEC) line (R-TNC.1) with nursing activity and thymocytes as well as BWRT 8 thymocyte hybridoma (TH) cells has been studied. The R-TNC.1 cell line significantly bound thymocytes and TH. Binding was stronger during the first 30 min of cell incubation and was followed by a progressive deadhesion. Among adherent thymocytes the proportion of apoptotic cells increased with culture time which was a consequence of higher capacity of the line for binding of apoptotic than viable cells and induction of apoptosis in a subset of adherent thymocytes. Emperiopolesis activity of this thymic nurse cell (TNC) line was manifested by engulfment of thymocytes as well as TH cells. A subset of viable intra-TNC thymocytes has been triggered to die by apoptosis, whereas other internalized thymocytes have been stimulated to proliferate, as measured by an increase in the percentage of cells in mitosis and higher incorporation of bromodeoxyuridine (BrdU), in comparison to thymocytes cultivated alone. A significant stimulation of proliferation of engulfed TH cells was also observed. The R-TNC.1 cell line efficiently phagocytosed both apoptotic thymocytes and TH, and the process is followed by intra-TNC destruction of ingested cells. Cumulatively, these results suggest different role of the R-TNC.1 clone: phagocytosis of apoptotic cells; induction of apoptotic cell death in a subset of both bound and internalized thymocytes and stimulation of proliferation of a subset of intra-TNC thymocytes or TH cells.

FIZZ1 and Ym as Tools to Discriminate between Differentially Activated Macrophages

Although it is well-established that macrophages can occur in distinct activation states, the molecular characteristics of differentially activated macrophages, and particularly those of alternatively activated macrophages (aaMφ), are still poorly unraveled. Recently, we demonstrated that the expression of FIZZ1 and Ym is induced in aaMφ as compared with classically activated macrophages (caMφ), elicitedin vitroor developedin vivoduring infection withTrypanosoma brucei brucei. In the present study, we analyzed the expression of FIZZ1 and Ym in caMφ and aaMφ elicited duringTrypanosoma congolenseinfection and show that the use of FIZZ1 and Ym for the identification of aaMφ is not limited toT. b. bruceiinfection and is independent of the organ sources from which macrophages are obtained. We also demonstrate that FIZZ1 can be used to discriminate between different populations of aaMφ. Furthermore, we studied the effects of various stimuli, and combinations thereof, on the expression of FIZZ1 and Ym in macrophages from different mouse strains and demonstrate that regulation of the expression of FIZZ1 and Ym in macrophages is not dependent on the mouse strain. Finally, we show that these genes can be used to monitor the macrophage activation status without the need to obtain pure macrophage populations.

Do Bovine Lymphocytes Express a Peculiar Prion Protein?

The cellular prion protein (PrPc) is a glycolipid-anchored cell surface protein that usually exhibits three glycosylation states. Its post-translationally modified isoform, PrPsc, is involved in the pathogenesis of various transmissible spongiform encephalopathies (TSEs). In bovine species, BSE infectivity appears to be restricted to the central nervous system; few or no detectable infectivity is found in lymphoid tissues in contrast to scrapie or variant CJD. Since expression of PrPc is a prerequisite for prion replication, we have investigated PrPc expression by bovine immune cells. Lymphocytes from blood and five different lymph organs were isolated from the same animal to assess intra- and interindividual variability of PrPc expression, considering six individuals. As shown by flow cytometry, this expression is absent or weak on granulocytes but is measurable on monocytes, B and T cells from blood and lymph organs. The activation of the bovine cells produces an upregulation of PrPc. The results of our in vitro study of PrPc biosynthesis are consistent with previous studies in other species. Interestingly, western blotting experiments showed only one form of the protein, the diglycosylated band. We propose that the glycosylation state could explain the lack of infectivity of the bovine immune cells.

Altered Affinity Maturation in Primary Response to (4-hydroxy-3-nitrophenyl) Acetyl (NP) after Autologous Reconstitution of Irradiated C57BL/6 Mice

Immune responses developing in irradiated environment are profoundly altered. The memory anti-arsonate response of A/J mice is dominated by a major clonotype encoded by a single gene segment combination called CRIA. In irradiated and autoreconstituted A/J mice, the level of anti-ARS antibodies upon secondary immunization is normal but devoid of CRIA antibodies. The affinity maturation process and the somatic mutation frequency are reduced. Isotype switching and development of germinal centers (GC) are delayed.The primary antibody response of C57BL/6 mice to the hapten (4-hydroxy-3-nitrophenyl) acetyl (NP)-Keyhole Limpet Hemocyanin (KLH) is dominated by antibodies encoded by a family of closely related VH genes associated with the expression of the λ1 light chain.We investigated the anti-NP primary response in irradiated and autoreconstituted C57BL/6 mice. We observed some splenic alterations as previously described in the irradiated A/J model. Germinal center reaction is delayed although the extrafollicular foci appearance is unchanged. Irradiated C57BL/6 mice are able to mount a primary anti-NP response dominated by λ1 positive antibodies but fail to produce high affinity NP-binding IgGl antibodies. Following a second antigenic challenge, irradiated mice develop enlarged GC and foci. Furthermore, higher affinity NP-binding IgG1 antibodies are detected.

Functional Tolerance is Maintained Despite Proliferation of CD4 T Cells after Encounter with Tissue-derived Antigen

Since negative selection in the thymus is incomplete, some self-reactive T cells are able to mature and seed the periphery. To study how these T cells interact following encounter with the self-protein they recognize in the periphery, we have developed an adoptive transfer system in which HEL-specific TCR transgenic CD4 T cells are transferred to mice expressing HEL protein in the pancreas under the control of the rat insulin promoter. Here we show that after adoptive transfer of HEL-specific T cells functional tolerance is maintained despite evidence that the T cells encounter and respond to pancreas-expressed antigen. Even the provision of an additional activation stimulus by peripheral immunization with HEL protein is insufficient to induce the T cells to cause autoimmune tissue injury. However, in the presence of blocking anti-CTLA-4-mAb, immunized adoptive transfer recipients rapidly developed diabetes. These data suggest that the CTLA-4 pathway regulates the pathogenicity of antigen-specific T cells following a peripheral activation stimulus.

Allergic Disease and Autoimmune Effectors Pathways

Allergy and autoimmunity result from dysregulation of the immune system. Until recently, it was generally accepted that the mechanisms that govern these disease processes are quite disparate; however, new discoveries suggest possible common pathogenetic effector pathways. This review illustrates the concomitant presentation of these conditions and the potential relationship or common mechanism in some cases, by looking at the key elements that regulate the immune response in both allergic and autoimmunite conditions: mast cells, antibodies, T cells, cytokines, and genetic determinants. The parallel appearance of allergic and autoimmune conditions in the some patients may reveal that such aberrations of the immune system have a common pathophysiologic mechanism. Mast cells, which play a key role in allergic reactions, and the wealth of inflammatory mediators they express, make it likely that they have profound effects on many autoimmune processes. Activation of protein kinases by inflammatory cytokines and environmental stresses may contribute to both allergic and autoimmune diseases. The presence of autoantibodies in some allergic conditions suggests an autoimmune basis for these conditions. Because of the central role T cells play in immune reactivity, the T-cell receptor (TCR) loci have long been considered important candidates for common disease susceptibility within the immune system such as asthma, atopy, and autoimmunity. Immunomodulation is the key to a successful treatment of allergic and autoimmune conditions.

Increased Frequency of Pre–Pro B Cells in the Bone Marrow of New Zealand Black (NZB) Mice: Implications for aDevelopmental Block in B Cell Differentiation

Reductions in populations of both Pre-B cell (Hardy fractions D) and Pro-B cells (Hardy fractions B–C) have been described in association with murine lupus. Recent studies of B cell populations, based on evaluation of B cell differentiation markers, now allow the enumeration and enrichment of other stage specific precursor cells. In this study we report detailed analysis of the ontogeny of B cell lineage subsets in New Zealand black (NZB) and control strains of mice. Our data suggest that B cell development in NZB mice is partially arrested at the fraction A Pre–Pro B cell stage. This arrest at the Pre-Pro B cell stage is secondary to prolonged lifespan and greater resistance to spontaneous apoptosis. In addition, expression of the gene encoding the critical B cell development transcription factor BSAP is reduced in the Pre–Pro B cell stage in NZB mice. This impairment may influence subsequent B cell development to later stages, and thereby accounts for the down-regulation of the B cell receptor componentIgα(mb-1). Furthermore, levels of expression of theRug2, λ5andIgβ(B29) genes are also reduced in Pre–Pro B cells of NZB mice. The decreased frequency of precursor B cells in the Pre–Pro B cell population occurs at the most primitive stage of B cell differentiation.

Elevated C-met in Thymic Dendritic Cells of New Zealand Black Mice

New Zealand Black (NZB) mice are a well-known animal model of human autoimmune disease. Although the mechanism for development of autoimmunity is unclear, NZB mice are well known for severe thymic microarchitecture abnormalities. It is thought that thymic dendritic cells (DC) may play a role in thymic education and contribute to the autoimmune process. To address this issue and, in particular, that qualitative and/or quantitative differences exist in thymic DC, we took advantage of a novel restriction analysis system that allow definition of differences in the expression of tyrosine kinases using highly enriched populations of thymic DC from NZB compared to BALB/c and C57BL/6 mice. The method chosen, restriction analysis of gene expression, allowed the determination of protein tyrosine kinase transcription profiles. We report herein that NZB mice have a significant upregulation of C-met compared to the control strains. The abnormality of the C-met transcription was confined to thymic DC. We believe that its abnormal expression reflects the resistance of thymic cells to apoptosis, which will ultimately lead to defects and/or abnormal signaling by the interaction of thymic DC and thymocytes. Further studies involving such interactions are under way.

Modulation of TNF-α Secretion in Peripheral Blood Mononuclear Cells by Cocoa Flavanols and Procyanidins

Epidemiological reports have suggested that the consumption of foods rich in flavonoids is associated with a lower incidence of certain degenerative diseases, including cardiovascular disease. Flavanols and their related oligomers, the procyanidins CFP, isolated from cocoa can modulate the production and level of several signaling molecules associated with immune function and inflammationin vitro, including several cytokines and eicosanoids. To further elucidate the potential immuno-modulatory functions of flavanol-rich cocoa, the present investigation examined whether isolated CFP fractions (monomers through decamers) influence the secretion of tumor necrosis factor-α (TNF-α) from resting and phytohemagluttinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC). We used anin vitroculture system where PBMC from 14 healthy subjects were introduced to individual CFP fractions for 72 h prior to measuring the levels of TNF-α released. The intermediate-sized CFP fractions (tetramers through octamers) were the most active on resting cells, causing a 3–4 fold increase in TNF-α relative to media baseline. The monomers and dimers were the least stimulatory of the fractions tested, displaying a 42 and 31% increase, respectively, over media control, whereas the trimers, nonamers and decamers showed an intermediate stimulation of this cytokine. In the presence of PHA, the intermediate-sized CFP fractions again were the most active, enhancing TNF-α secretion in the range of 48–128% relative to the PHA control. The monomers and dimers were slightly inhibitory (–1.5 and –15%, respectively), while trimers, nonamers and decamers stimulated moderate increases in TNF-α levels (13, 19 and 15%, respectively). The above results lend support to the concept that CFP can be immunomodulatory. The stimulation of TNF-α secretion may contribute to the putative beneficial effects of dietary flavanoids against microbial infection and tumorigenesis.