Receptor editing constrains development of phosphatidyl choline-specific B cells in VH12-transgenic mice.

B1 B cells reactive to phosphatidyl choline (PtC) exhibit restricted immunoglobulin heavy chain (HC) and light chain (LC) combinations, exemplified by VH12/Vκ4/5H. Two checkpoints are thought to focus PtC+ B cell maturation in VH12-transgenic mice (VH12 mice): V-J rearrangements encoding a "permissive" LC capable of VH12 HC pairing are selected first, followed by positive selection based on PtC binding, often requiring LC receptor editing to salvage PtC- B cells and acquire PtC reactivity. However, evidence obtained from breeding VH12 mice to editing-defective dnRAG1 mice and analyzing LC sequences from PtC+ and PtC- B cell subsets instead suggests that receptor editing functions after initial positive selection to remove PtC+ B cells in VH12 mice. This offers a mechanism to constrain natural, polyreactive B cells to limit their frequency. Sequencing also reveals occasional in-frame hybrid LC genes, reminiscent of type 2 gene replacement, that, testing suggests, arise via a recombination-activating gene (RAG)-independent mechanism.

Erythrosin B: a versatile colorimetric and fluorescent vital dye for bacteria.

Rapidly assaying cell viability for diverse bacteria species is not always straightforward. In eukaryotes, cell viability is often determined using colorimetric dyes; however, such dyes have not been identified for bacteria. We screened different dyes and found that erythrosin B (EB), a visibly red dye with fluorescent properties, functions as a vital dye for many Gram-positive and -negative bacteria. EB worked at a similar concentration for all bacteria studied and incubations were as short as 5 min. Given EB's spectral properties, diverse experimental approaches are possible to rapidly visualize and/or quantitate dead bacterial cells in a population. As the first broadly applicable colorimetric viability dye for bacteria, EB provides a cost-effective alternative for researchers in academia and industry.

IL10 restrains autoreactive B cells in transgenic mice expressing inactive RAG1.

IL10 plays a dual role in supporting humoral immunity and inhibiting inflammatory conditions. B cells producing IL10 are thought to play a key regulatory role in maintaining self-tolerance and suppressing excessive inflammation during autoimmune and infectious diseases, primarily by inhibiting associated T cell responses. The extent to which B cells, through the provision of IL10, might function to sustain or inhibit autoantibody production is less clear. We previously described transgenic mice expressing catalytically inactive RAG1 (dnRAG1 mice), which show expansion of an IL10-compentent CD5+ B cell subset that phenotypically resembles B10 B cells, hypogammaglobulinemia, and a restricted B cell receptor repertoire with features indicative of impaired B cell receptor editing. We show here that B10-like B cells in dnRAG1 mice bind the membrane-associated autoantigen phosphatidylcholine (PtC), and that in vitro lipopolysaccharide (LPS) stimulation of dnRAG1 splenocytes induces a robust IgM response enriched in reactivity toward lupus-associated autoantigens. This outcome was correlated with detection of sIgMhi B cell populations that were distinct from, but in addition to, sIgMint populations observed after similar treatment of wild-type splenocytes. Loss of IL10 expression in dnRAG1 mice had no significant effect on B10-like B cell expansion or the frequency of PtC+ B cells. Compared to IL10+/+ dnRAG1 mice, levels of serum IgM, but not serum IgG, were highly elevated in some naïve IL10-/- dnRAG1 mice, and was correlated with a significant increase in serum BAFF levels. Differentiation of sIgMint B cells from LPS-stimulated dnRAG1 splenocytes was enhanced by loss of IL10 expression and IL10 blockade, but was suppressed by treatment with recombinant IL10. In vitro LPS-induced differentiation and antibody production was inhibited by treatment with JAK/STAT inhibitors or a synthetic corticosteroid, independent of IL10 expression and genotype. Taken together, these data suggest that IL10 expression in dnRAG1 mice maintains suppression of IgM levels in part by inhibiting BAFF production, and that regulatory B10-like B cells, through the provision of IL10, constrains B cell differentiation in response to mitogenic stimuli. Furthermore, autoantibody profiling raises a possible link between CD5+ B cell expansion, mitogenic stimulation, and autoantibodies associated with autoimmune complications observed in lupus and lupus-related disorders.

VprBP (DCAF1) Regulates RAG1 Expression Independently of Dicer by Mediating RAG1 Degradation.

The assembly of Ig genes in developing B lymphocytes by V(D)J recombination is initiated by the RAG1-RAG2 endonuclease complex. We previously identified an interaction between RAG1 and viral protein R binding protein (VprBP) (also known as DNA damage binding protein 1 cullin 4-associated factor 1 [DCAF1]), a substrate receptor for the cullin 4-really interesting new gene (RING) E3 ubiquitin ligase (CRL4). We report in this article that in mice, B cell-intrinsic loss of VprBP increases RAG1 protein levels and disrupts expression of the endoribonuclease Dicer, which is essential for microRNA maturation. Rag1/2 transcription is known to be derepressed by loss of microRNA-mediated suppression of phosphatase and tensin homolog, raising the possibility that the elevated level of RAG1 observed in VprBP-deficient B cells is caused indirectly by the loss of Dicer. However, we show that VprBP restrains RAG1 expression posttranscriptionally and independently of Dicer. Specifically, loss of VprBP stabilizes RAG1 protein, which we show is normally degraded via a mechanism requiring both 20S proteasome and cullin-RING E3 ubiquitin ligase activity. Furthermore, we show that RAG1 stabilization through small molecule inhibition of cullin-RING E3 ubiquitin ligase activation promotes V(D)J recombination in a murine pre-B cell line. Thus, in addition to identifying a role for VprBP in maintaining Dicer levels in B cells, our findings reveal the basis for RAG1 turnover and provide evidence that the CRL4VprBP(DCAF1) complex functions to maintain physiological levels of V(D)J recombination.

Cd1d regulates B cell development but not B cell accumulation and IL10 production in mice with pathologic CD5(+) B cell expansion.

BACKGROUND: CD1d is a widely expressed lipid antigen presenting molecule required for CD1d-restricted invariant natural killer T (iNKT) cell development. Elevated CD1d expression is detected in CD5(+) IL10-producing B cells, called B10 B cells, and is correlated with poorer prognosis in chronic lymphocytic leukemia (CLL), a CD5(+) B cell malignancy with B10-like functional properties. Whether CD1d expression regulates CD5(+) B cell accumulation, IL10 competence, and antibody production in naïve mice with pathologic CD5(+) B cell expansion remains untested. RESULTS: Using three different transgenic mouse models of benign or leukemic CD5(+) B cell expansion, we found that CD1d was differentially expressed on CD5(+) B cells between the three models, but loss of CD1d expression had no effect on CD5(+) B cell abundance or inducible IL10 expression in any of the models. Interestingly, in the CLL-prone Eµ-TCL1 model, loss of CD1d expression suppressed spontaneous IgG (but not IgM) production, whereas in the dnRAG1xEµ-TCL1 (DTG) model of accelerated CLL, loss of CD1d expression was associated with elevated numbers of splenic CD4(+) and CD8(+) T cells and an inverted CD4(+):CD8(+) T cell ratio. Unexpectedly, before leukemia onset, all three transgenic CD1d-deficient mouse strains had fewer splenic transitional B cells than their CD1d-proficient counterparts. CONCLUSIONS: The results show that CD1d expression and iNKT cells are dispensable for the development, accumulation, or IL10 competence of CD5(+) B cells in mice prone to benign or leukemic CLL-like B cell expansion, but reveal a novel role for iNKT cells in supporting B cell progression through the transitional stage of development in these animals. These results suggest CD1d-directed therapies to target CLL could be evaded by downregulating CD1d expression with little effect on continued leukemic CD5(+) B cell survival. The data also imply that iNKT cells help restrain pro-leukemic CD8(+) T cell expansion in CLL, potentially explaining a reported correlation in human CLL between disease progression, the loss of NKT cells, and a paradoxical increase in CD8(+) T cells.

VprBP Is Required for Efficient Editing and Selection of Igκ+ B Cells, but Is Dispensable for Igλ+ and Marginal Zone B Cell Maturation and Selection.

B cell development past the pro-B cell stage in mice requires the Cul4-Roc1-DDB1 E3 ubiquitin ligase substrate recognition subunit VprBP. Enforced Bcl2 expression overcomes defects in distal VH-DJH and secondary Vκ-Jκ rearrangement associated with VprBP insufficiency in B cells and substantially rescues maturation of marginal zone and Igλ(+) B cells, but not Igκ(+) B cells. In this background, expression of a site-directed Igκ L chain transgene increases Igκ(+) B cell frequency, suggesting VprBP does not regulate L chain expression from a productively rearranged Igk allele. In site-directed anti-dsDNA H chain transgenic mice, loss of VprBP function in B cells impairs selection of Igκ editor L chains typically arising through secondary Igk rearrangement, but not selection of Igλ editor L chains. Both H and L chain site-directed transgenic mice show increased B cell anergy when VprBP is inactivated in B cells. Taken together, these data argue that VprBP is required for the efficient receptor editing and selection of Igκ(+) B cells, but is largely dispensable for Igλ(+) B cell development and selection, and that VprBP is necessary to rescue autoreactive B cells from anergy induction.

Accelerated progression of chronic lymphocytic leukemia in Eµ-TCL1 mice expressing catalytically inactive RAG1.

Chronic lymphocytic leukemia (CLL) is a prevalent B-cell neoplasia that is often preceded by a more benign monoclonal CD5(+) B-cell lymphocytosis. We previously generated transgenic mice expressing catalytically inactive RAG1 (dominant-negative recombination activating gene 1 [dnRAG1] mice) that develop an early-onset indolent CD5(+) B-cell lymphocytosis attributed to a defect in secondary V(D)J rearrangements initiated to edit autoreactive B-cell receptor (BCR) specificity. Hypothesizing that CD5(+) B cells in these animals represent potential CLL precursors, we crossed dnRAG1 mice with CLL-prone Eµ-TCL1 mice to determine whether dnRAG1 expression in Eµ-TCL1 mice accelerates CLL onset. Consistent with this hypothesis, CD5(+) B-cell expansion and CLL progression occurred more rapidly in double-transgenic mice compared with Eµ-TCL1 mice. Nevertheless, CD5(+) B cells in the 2 mouse strains exhibited close similarities in phenotype, immunoglobulin gene usage, and mutation status, and expression of genes associated with immune tolerance and BCR signaling. Gene expression profiling further revealed a potential role for prolactin signaling in regulating BCR editing. These results suggest a model in which benign accumulation of CD5(+) B cells can be initiated through a failure to successfully edit autoreactive BCR specificity and may, in turn, progress to CLL upon introduction of additional genetic mutations.

VprBP binds full-length RAG1 and is required for B-cell development and V(D)J recombination fidelity.

The N-terminus of full-length RAG1, though dispensable for RAG1/2 cleavage activity, is required for efficient V(D)J recombination. This region supports RING E3 ubiquitin ligase activity in vitro, but whether full-length RAG1 functions as a single subunit or a multi-subunit E3 ligase in vivo is unclear. We show the multi-subunit cullin RING E3 ligase complex VprBP/DDB1/Cul4A/Roc1 associates with full-length RAG1 through VprBP. This complex is assembled into RAG protein-DNA complexes, and supports in-vitro ubiquitylation activity that is insensitive to RAG1 RING domain mutations. Conditional B lineage-specific VprBP disruption arrests B-cell development at the pro-B-to-pre-B cell transition, but this block is bypassed by expressing rearranged immunoglobulin transgenes. Mice with a conditional VprBP disruption show modest reduction of D-J(H) rearrangement, whereas V(H)-DJ(H) and V(κ)-J(κ) rearrangements are severely impaired. D-J(H) coding joints from VprBP-insufficent mice show longer junctional nucleotide insertions and a higher mutation frequency in D and J segments than normal. These data suggest full-length RAG1 recruits a cullin RING E3 ligase complex to ubiquitylate an unknown protein(s) to limit error-prone repair during V(D)J recombination.

Accumulation of B1-like B cells in transgenic mice over-expressing catalytically inactive RAG1 in the periphery.

During their development, B lymphocytes undergo V(D)J recombination events and selection processes that, if successfully completed, produce mature B cells expressing a non-self-reactive B-cell receptor (BCR). Primary V(D)J rearrangements yield self-reactive B cells at high frequency, triggering attempts to remove, silence, or reprogramme them through deletion, anergy induction, or secondary V(D)J recombination (receptor editing), respectively. In principle, expressing a catalytically inactive V(D)J recombinase during a developmental stage in which V(D)J rearrangement is initiated may impair this process. To test this idea, we generated transgenic mice expressing a RAG1 active site mutant (dnRAG1 mice); RAG1 transcript was elevated in splenic, but not bone marrow, B cells in dnRAG1 mice relative to wild-type mice. The dnRAG1 mice accumulate splenic B cells with a B1-like phenotype that exhibit defects in B-cell activation, and are clonally diverse, yet repertoire restricted with a bias toward Jκ1 gene segment usage. The dnRAG1 mice show evidence of impaired B-cell development at the immature-to-mature transition, immunoglobulin deficiency, and poorer immune responses to thymus-independent antigens. Interestingly, dnRAG1 mice expressing the anti-dsDNA 3H9H56R heavy chain fail to accumulate splenic B1-like cells, yet retain peritoneal B1 cells. Instead, these mice show an expanded marginal zone compartment, but no difference is detected in the frequency of heavy chain gene replacement. Taken together, these data suggest a model in which dnRAG1 expression impairs secondary V(D)J recombination. As a result, selection and/or differentiation processes are altered in a way that promotes expansion of B1-like B cells in the spleen.

Collagen XIII induced in vascular endothelium mediates alpha1beta1 integrin-dependent transmigration of monocytes in renal fibrosis.

Alport syndrome is a common hereditary basement membrane disorder caused by mutations in the collagen IV α3, α4, or α5 genes that results in progressive glomerular and interstitial renal disease. Interstitial monocytes that accumulate in the renal cortex from Alport mice are immunopositive for integrin α1ß1, while only a small fraction of circulating monocytes are immunopositive for this integrin. We surmised that such a disparity might be due to the selective recruitment of α1ß1-positive monocytes. In this study, we report the identification of collagen XIII as a ligand that facilitates this selective recruitment of α1ß1 integrin-positive monocytes. Collagen XIII is absent in the vascular endothelium from normal renal cortex and abundant in Alport renal cortex. Neutralizing antibodies against the binding site in collagen XIII for α1ß1 integrin selectively block VLA1-positive monocyte migration in transwell assays. Injection of these antibodies into Alport mice slows monocyte recruitment and protects against renal fibrosis. Thus, the induction of collagen XIII in endothelial cells of Alport kidneys mediates the selective recruitment of α1ß1 integrin-positive monocytes and may potentially serve as a therapeutic target for inflammatory diseases in which lymphocyte/monocyte recruitment involves the interaction with α1ß1 integrin.

Cigarette smoke-induced effects on bone marrow B-cell subsets and CD4+:CD8+ T-cell ratios are reversed by smoking cessation: influence of bone mass on immune cell response to and recovery from smoke exposure.

Cigarette smoking adversely affects the immune system, and is a risk factor for developing osteoporosis. How smoking contributes to osteoporosis is unclear, but since lymphocytes help maintain bone homeostasis and lymphocyte depletion results in bone loss, one potential mechanism for how smoke exposure promotes osteoporosis is by reducing bone marrow lymphocytes. Since the risk for developing osteoporosis is reportedly greater in smokers with polymorphisms in LRP5, a gene involved in canonical Wnt signaling that regulates bone metabolism, smoking-induced effects on lymphocytes may be influenced by Lrp5 functionality. To test these possibilities, we examined how the duration and cessation of cigarette smoke exposure affects lymphocyte distribution and function in normal mice and mice predisposed to low or high bone mass due to disruption or mutation of Lrp5. We find that, independent of genotype, mice exposed to cigarette smoke for 3-12 weeks showed a significant reduction in bone marrow B220(+)CD43(-) B cells and splenic transitional T1 B cells, and exhibited a splenic CD4(+):CD8(+) T-cell ratio that was skewed toward CD8(+) T cells. Smoke exposure had little or no effect on other lymphocyte subsets or on lymphocyte function ex vivo. Interestingly, these differences were no longer apparent after 6 weeks without smoke exposure, except in mice with high bone mass where bone marrow B220(+)CD43(-) B cells failed to fully recover. These data provide the first evidence that smoke exposure reduces bone marrow B cells, providing a plausible mechanism for how smoking contributes to osteoporosis.

A flow cytometric assay to quantify in vivo bacterial uptake by alveolar macrophages.

Our laboratory has developed a flow cytometric assay to quantify alveolar macrophage (Mcapital EF, Cyrillic) phagocytosis of bacteria within a live animal. Mcapital EF, Cyrillics collected by bronchoalveolar lavage from rats infected transtracheally with Syto 9-labeled bacteria are fluorescently labeled for identification and analyzed by flow cytometry to quantify their bacterial uptake.

Lysophosphatidic Acid signals through specific lysophosphatidic Acid receptor subtypes to control key regenerative responses of human gingival and periodontal ligament fibroblasts.

BACKGROUND: We showed that the pluripotent platelet growth factor and mediator lysophosphatidic acid (LPA) controls key regenerative responses of human gingival fibroblasts (GFs) and periodontal ligament fibroblasts (PDLFs) and positively modulates their responses to platelet-derived growth factor (PDGF). This study determined which LPA receptor (LPAR) subtype(s) LPA signals through to stimulate mitogenic extracellular signal-regulated kinase (ERK) 1/2 signaling and chemotaxis and to elicit intracellular Ca(2+) increases in GFs and PDLFs because many healing responses are calcium-dependent. METHODS: Activation of mitogen-activated protein kinase was determined using Western blotting with an antibody to phosphorylated ERK1/2. Migration responses were measured using a microchemotaxis chamber. GF and PDLF intracellular Ca(2+) mobilization responses to multiple LPA species and LPAR subtype-specific agonists were measured by using a cell-permeable fluorescent Ca(2+) indicator dye. RESULTS: LPA stimulated ERK1/2 phosphorylation via LPA(1)(-3). For GFs, LPA(1) preferentially elicited chemotaxis, and LPA(1-3) for PDLFs, as confirmed using subtype-specific agonists. Elevation of intracellular calcium seems to be mediated through LPA(1) and LPA(3), with little, if any, contribution from LPA(2). CONCLUSIONS: To the best of our knowledge, this study provides the first evidence that LPA signals through specific LPAR subtypes to stimulate human oral fibroblast regenerative responses. These data, in conjunction with our previous findings showing that LPA modulates GF and PDLF responses to PDGF, suggest that LPA is a factor of emerging importance to oral wound healing.

Smoke exposure and ethanol ingestion modulate intrapulmonary polymorphonuclear leukocyte killing, but not recruitment or phagocytosis.

BACKGROUND: People who smoke and abuse alcohol are uniquely susceptible to pulmonary infections caused by Streptococcus pneumoniae, the pneumococcus. The primary cellular defense against pneumococci within the lungs is the polymorphonuclear leukocyte (PMN). Cigarette smoke and ethanol (EtOH) are known to alter certain PMN functions, but little is known about their concurrent effects. METHODS: Male Sprague-Dawley rats were exposed twice daily for 8 weeks to cigarette smoke (smoke-exposed) or room air (sham-exposed). During the final week of exposure, the rats were pair-fed a liquid diet containing either 36 or 0% EtOH calories. Polymorphonuclear leukocytes were prerecruited into the rats' lungs by transtracheal injection of lipopolysaccharide. Five hours later, the rats were infected transtracheally with S. pneumoniae, and PMN recruitment, phagocytosis, and bactericidal activity were quantified within their lungs. Chemokine levels were also measured in bronchoalveolar lavage fluids, lung homogenates, and sera. RESULTS: Neither PMN recruitment nor phagocytic uptake of pneumococci was altered by EtOH ingestion or smoke exposure. Killing of the organisms, however, was significantly decreased in sham-exposed, but not smoke-exposed, rats ingesting EtOH. Parallel results were determined for serum cytokine-induced neutrophil chemoattractant-1 (CINC-1), with EtOH ingestion significantly decreasing the levels in sham-exposed, but not smoke-exposed, rats. Pulmonary levels of macrophage inflammatory protein-2 (MIP-2) and CINC-1 were highly elevated by the combination of EtOH and smoke. CONCLUSIONS: One week of EtOH ingestion by rats impaired the ability of their PMNs to kill S. pneumoniae within their lungs. This was not due to decreased recruitment of the PMNs to the lungs or to diminished phagocytosis of intrapulmonary pneumococci. The addition of twice-daily cigarette smoke exposure to this short-term EtOH ingestion model restored PMN bactericidal ability to levels observed in the absence of either treatment. These EtOH-induced and smoke-induced alterations in PMN killing may be related to alterations in both pulmonary and systemic inflammatory chemokine levels.

A novel flow cytometric assay for measurement of in vivo pulmonary neutrophil phagocytosis.

BACKGROUND: Phagocytosis assays are traditionally performed in vitro using polymorphonuclear leukocytes (PMNs) isolated from peripheral blood or the peritoneum and heat-killed, pre-opsonized organisms. These assays may not adequately mimic the environment within the infected lung. Our laboratory therefore has developed a flow cytometric in vivo phagocytosis assay that enables quantification of PMN phagocytosis of viable bacteria within the lungs of rats. In these studies, rats are injected transtracheally with lipopolysaccharide (LPS) to recruit PMNs to their lungs. They are then infected with live 5(-and 6) carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) labeled type 3 Streptococcus pneumoniae. Bronchoalveolar lavage is performed and resident alveolar macrophages and recruited PMNs are labeled with monoclonal antibodies specific for surface epitopes on each cell type. Three color flow cytometry is utilized to identify the cell types, quantify recruitment, and determine uptake of the labeled bacteria. RESULTS: The viability of the alveolar macrophages and PMNs isolated from the lavage fluid was >95%. The values of the percentage of PMNs in the lavage fluid as well as the percentage of PMNs associated with CFSE-labeled S. pneumoniae as measured through flow cytometry showed a high degree of correlation with the results from manual counting of cytospin slides. CONCLUSION: This assay is suitable for measuring bacterial uptake within the infected lung. It can be adapted for use with other organisms and/or animal model systems.

Increased expression and activation of CD30 induce apoptosis in human blood eosinophils.

Eosinophils are one of the major effector cells in asthma, and controlling the number and survival of eosinophils might attenuate the severity of asthma. This result could be achieved by inducing eosinophil apoptosis. Apoptosis allows the removal of cells without inducing an inflammatory response. Our knowledge of the factors involved in regulating eosinophil apoptosis remains limited. CD30 molecule has been associated with T cell-negative selection and in TCR-mediated apoptosis. In this study we examined the expression and role of CD30 in apoptosis of human blood eosinophils. Percentage of apoptotic eosinophils was determined by annexin V-propidium iodide labeling, and CD30 expression was examined by flow cytometry. Spontaneous apoptosis was induced by serum deprivation, and survival was conferred by incubating cells with 10% FBS and IL-5. CD30 surface expression was up-regulated in eosinophils incubated for 24 h as compared with freshly isolated eosinophils, and both CD30 expression and eosinophil apoptosis increased in a time-dependent manner. We also measured CD30 mRNA expression by quantitative real-time RT-PCR and determined that CD30 transcripts increased in eosinophils undergoing apoptosis only under serum deprivation conditions. The agonistic CD30 Abs, Ber-H8 and HeFi-1, significantly enhanced eosinophil apoptosis. FBS and IL-5 failed to inhibit or suppress the CD30 agonistic-induced apoptosis. These data support the role of CD30 activation in eosinophil apoptosis. This research will help in furthering our understanding of eosinophil apoptosis and therefore might contribute to the development of better therapeutic modalities in the treatment and/or cure of allergic inflammation in bronchial asthma.