T cell-derived interferon-γ programs stem cell death in immune-mediated intestinal damage.

Despite the importance of intestinal stem cells (ISCs) for epithelial maintenance, there is limited understanding of how immune-mediated damage affects ISCs and their niche. We found that stem cell compartment injury is a shared feature of both alloreactive and autoreactive intestinal immunopathology, reducing ISCs and impairing their recovery in T cell-mediated injury models. Although imaging revealed few T cells near the stem cell compartment in healthy mice, donor T cells infiltrating the intestinal mucosa after allogeneic bone marrow transplantation (BMT) primarily localized to the crypt region lamina propria. Further modeling with ex vivo epithelial cultures indicated ISC depletion and impaired human as well as murine organoid survival upon coculture with activated T cells, and screening of effector pathways identified interferon-γ (IFNγ) as a principal mediator of ISC compartment damage. IFNγ induced JAK1- and STAT1-dependent toxicity, initiating a proapoptotic gene expression program and stem cell death. BMT with IFNγ-deficient donor T cells, with recipients lacking the IFNγ receptor (IFNγR) specifically in the intestinal epithelium, and with pharmacologic inhibition of JAK signaling all resulted in protection of the stem cell compartment. In addition, epithelial cultures with Paneth cell-deficient organoids, IFNγR-deficient Paneth cells, IFNγR-deficient ISCs, and purified stem cell colonies all indicated direct targeting of the ISCs that was not dependent on injury to the Paneth cell niche. Dysregulated T cell activation and IFNγ production are thus potent mediators of ISC injury, and blockade of JAK/STAT signaling within target tissue stem cells can prevent this T cell-mediated pathology.

NKT cells in mucosal immunity.

The gastrointestinal tract allows the residence of an almost enumerable number of bacteria. To maintain homeostasis, the mucosal immune system must remain tolerant to the commensal microbiota and eradicate pathogenic bacteria. Aberrant interactions between the mucosal immune cells and the microbiota have been implicated in the pathogenesis of inflammatory disorders, such as inflammatory bowel disease (IBD). In this review, we discuss the role of natural killer T cells (NKT cells) in intestinal immunology. NKT cells are a subset of non-conventional T cells recognizing endogenous and/or exogenous glycolipid antigens when presented by the major histocompatibility complex (MHC) class I-like antigen-presenting molecules CD1d and MR1. Upon T-cell receptor (TCR) engagement, NKT cells can rapidly produce various cytokines that have important roles in mucosal immunity. Our understanding of NKT-cell-mediated pathways including the identification of specific antigens is expanding. This knowledge will facilitate the development of NKT cell-based interventions and immune therapies for human intestinal diseases.

Bruton's tyrosine kinase regulates the activation of gene rearrangements at the lambda light chain locus in precursor B cells in the mouse.

Bruton's tyrosine kinase (Btk) is a nonreceptor tyrosine kinase involved in precursor B (pre-B) cell receptor signaling. Here we demonstrate that Btk-deficient mice have an approximately 50% reduction in the frequency of immunoglobulin (Ig) lambda light chain expression, already at the immature B cell stage in the bone marrow. Conversely, transgenic mice expressing the activated mutant Btk(E41K) showed increased lambda usage. As the kappa/lambda ratio is dependent on (a) the level and kinetics of kappa and lambda locus activation, (b) the life span of pre-B cells, and (c) the extent of receptor editing, we analyzed the role of Btk in these processes. Enforced expression of the Bcl-2 apoptosis inhibitor did not alter the Btk dependence of lambda usage. Crossing 3-83mudelta autoantibody transgenic mice into Btk-deficient mice showed that Btk is not essential for receptor editing. Also, Btk-deficient surface Ig(+) B cells that were generated in vitro in interleukin 7-driven bone marrow cultures manifested reduced lambda usage. An intrinsic defect in lambda locus recombination was further supported by the finding in Btk-deficient mice of reduced lambda usage in the fraction of pre-B cells that express light chains in their cytoplasm. These results implicate Btk in the regulation of the activation of the lambda locus for V(D)J recombination in pre-B cells.

Cation- and peptide-binding properties of human centrin 2.

Centrin and calmodulin (CaM) are closely related four-EF-hand Ca(2+)-binding proteins. While CaM is monomeric, centrin 2 is dimeric and binds only two Ca(2+) per dimer, likely to site IV in each monomer. Ca(2+) binding to centrin 2 displays pronounced negative cooperativity and a [Ca(2+)](0.5) of 30 microM. As in CaM, Ca(2+) binding leads to the exposure of a hydrophobic probe-accessible patch on the surface of centrin 2. Provided Ca(2+) is present, centrin 2 forms a 1:1 peptide:monomer complex with melittin with an affinity of 100 nM. The complex binds four instead of two Ca(2+). Our data point to surprising differences in the mode of activation of these homologous proteins.

Differential expression and cellular distribution of centrin isoforms during human ciliated cell differentiation in vitro.

Centrin protein is an ubiquitously expressed cytoskeletal component and is a member of the EF-hand superfamily of calcium-binding proteins. It was first discovered in the flagellar apparatus of unicellular green algae where it is involved in contraction of Ca(2+)-sensitive structures. Centrin protein is associated with centrosome-related structures such as spindle pole body in yeast, and centriole/basal bodies in flagellar and ciliated cells. Three centrin genes have been cloned in human cells. In this work, we have performed a comparative biochemical and functional analysis of centrin isoforms using a primary culture of human nasal epithelial cells which provides an efficient way to obtain a complete ciliated cell differentiation process. RT-PCR experiments show that the expression of the three human centrin genes increases during cell differentiation, and that only centrin 2 and 3 are expressed during cell proliferation. Using polyclonal antibodies raised against recombinant human centrin 2 and 3, we show a specific pattern of protein expression. Ultrastructural immunolocalization suggests that centrin proteins are involved in the early process of centriole assembly, as they are concentrated within the precursor structures of centriole/basal bodies. It also shows a differential localisation of centrin proteins in mature centriole/basal bodies, suggesting different functions for centrins 1/2 and centrin 3. This is also supported by functional analyses showing that centrin 1 and/or centrin 2 are involved in ciliary beating.

A role for centrin 3 in centrosome reproduction.

Centrosome reproduction by duplication is essential for the bipolarity of cell division, but the molecular basis of this process is still unknown. Mutations in Saccharomyces cerevisiae CDC31 gene prevent the duplication of the spindle pole body (SPB). The product of this gene belongs to the calmodulin super-family and is concentrated at the half bridge of the SPB. We present a functional analysis of HsCEN3, a human centrin gene closely related to the CDC31 gene. Transient overexpression of wild-type or mutant forms of HsCen3p in human cells demonstrates that centriole localization depends on a functional fourth EF-hand, but does not produce mitotic phenotype. However, injection of recombinant HsCen3p or of RNA encoding HsCen3p in one blastomere of two-cell stage Xenopus laevis embryos resulted in undercleavage and inhibition of centrosome duplication. Furthermore, HsCEN3 does not complement mutations or deletion of CDC31 in S. cerevisiae, but specifically blocks SPB duplication, indicating that the human protein acts as a dominant negative mutant of CDC31. Several lines of evidence indicate that HsCen3p acts by titrating Cdc31p-binding protein(s). Our results demonstrate that, in spite of the large differences in centrosome structure among widely divergent species, the centrosome pathway of reproduction is conserved.

A pollen model in the rat for testing adjuvant activity of air pollution components.

During the last decades, the prevalence of allergy has increased worldwide. Allergic rhinitis ("hay fever") and asthma are two of the most common allergic diseases. A possible cause for increased allergy to pollen is air pollution. The increase of industrialization and the number of diesel engines associated with diesel exhaust particles (DEP) in the air parallel the increase in allergic airway diseases. To investigate the adjuvant effect of DEP in pollen allergy, Brown Norway (BN) rats were sensitized intranasally or intratracheally with timothy grass pollen (Phleum pratense) with or without DEP (3 mg/ml). Intranasal sensitization (200 microl, 10 mg/ml) was performed daily for 5 consecutive days and intratracheal sensitization (200 microl, 10 mg/ml) was performed once. Challenge with pollen was performed at day 21 similarly to the sensitization protocol. Blood samples were taken at day 28 after the first sensitization. The binding of DEP to pollen grains was studied by scanning electron microscopy and the inflammatory response in the lung was studied by light microscopy. Immunoglobulin E (IgE) and IgG(1) responses against pollen grains were measured by digoxigenin (DIG) enzyme-linked immunosorbent assay (ELISA). Scanning electron microscopy revealed a mixture of free DEP and DEP associated with pollen grains. Both intranasal and intratracheal routes of administration of pollen grains induced inflammatory reactions in the lung with an influx of macrophages, eosinophilic granulocytes, and granuloma formation. Pollen grains were localized in the alveoli after both intranasal and intratracheal administration and were surrounded by macrophages. The number and localization of pollen grains were similar for both routes of administration. After coexposure with DEP, DEP-loaded macrophages were found around the pollen. Localization, inflammatory reaction, and integrity of pollen were similar to those seen without DEP. At day 28, specific IgE and IgG(1) antibodies were found in serum of rats immunized intranasally or intratracheally. IgE antibody response was higher in rats immunized with pollen grains and DEP than in rats immunized with pollen only (dilution mean +/- SEM: 59.4 +/- 4.6 vs. 27 +/- 5.1). The IgG(1) antibody response was much higher compared to the IgE response (factor of 10(4)), but the level of IgG(1) antibodies was only slightly increased by DEP (dilution mean +/- SEM: 24.2 +/- 2.0 x 10(4) vs. 16.1 +/- 2.1 x 10(4)). In conclusion, the intranasal application of pollen in the BN rat is a suitable and elegant method to evoke inflammatory reactions in the lung and pollen-specific IgE responses measured by DIG ELISA. Finally, this model gives similar results on adjuvant activity of DEP found in the ovalbumin models presented previously.

Identification of a new mammalian centrin gene, more closely related to Saccharomyces cerevisiae CDC31 gene.

Among the numerous centrin isoforms identified by two-dimensional gel electrophoresis in human cells, an acidic and slow-migrating isoform is particularly enriched in a centrosome fraction. We report here that this isoform specifically reacts with antibodies raised against Saccharomyces cerevisiae Cdc31p and is present, as other centrin isoforms, in the distal lumen of centrioles. It is encoded by a new centrin gene, which we propose to name HsCEN3 (Homo sapiens centrin gene 3). This gene is more closely related to the yeast CDC31 gene, and shares less identity with algae centrin than HsCEN1 and HsCEN2. A murine CDC31-related gene was also found that shows 98% identity and 100% similarity with HsCEN3, demonstrating a higher interspecies conservation than the murine centrin gene MmCEN1 (Mus musculus centrin gene 1) with either HsCEN1, or HsCEN2. Finally, immunological data suggest that a CDC31-related gene could exist in amphibians and echinoderms as well. All together, our data suggest the existence of two divergent protein subfamilies in the current centrin family, which might be involved in distinct centrosome-associated functions. The possible implication of this new mammalian centrin gene in centrosome duplication is discussed.

Anchorage-dependent transcription of the cyclin A gene.

NIH 3T3 cells cultured in suspension fail to express cyclin A and hence cannot enter S phase and divide. We show that loss of cell adhesion to substratum abrogates cyclin A gene expression by blocking its promoter activity through the E2F site that mediates its cell cycle regulation in adherent cells. In suspended cells, G0-specific E2F complexes remain bound to the cyclin A promoter. Overexpression of cyclin D1 restores cyclin A transcription in suspended cells and rescues them from cell cycle arrest. In suspended cells, cyclin D1 and cyclin E accumulate normally upon serum stimulation, but their associated kinases remain inactive; their substrates, pRb and p107, are not hyperphosphorylated. Concomitantly, the cyclin-dependent kinase inhibitor, p27KIP1, is stabilized. Ectopic expression of p27KIP1 blocks cyclin A promoter activity through its EN binding site. These data suggest that the block to cyclin A transcription in nonadherent NIH 3T3 cells results from stabilization of p27KIP1 and subsequent inactivation of the specific E2F moiety required for its induction.

Cell cycle regulation of the cyclin A gene promoter is mediated by a variant E2F site.

Cyclin A is involved in the control of S phase and mitosis in mammalian cells. Expression of the cyclin A gene in nontransformed cells is characterized by repression of its promoter during the G1 phase of the cell cycle and its induction at S-phase entry. We show that this mode of regulation is mediated by the transcription factor E2F, which binds to a specific site in the cyclin A promoter. It differs from the prototype E2F site in nucleotide sequence and protein binding; it is bound by E2F complexes containing cyclin E and p107 but not pRB. Ectopic expression of cyclin D1 triggers premature activation of the cyclin A promoter by E2F, and this effect is blocked by the tumor suppressor protein p16INK4.