Dendritic cells from lupus-prone mice are defective in repressing immunoglobulin secretion

Autoimmunity results from a breakdown in tolerance mechanisms that regulate autoreactive lymphocytes. We recently showed that during innate immune responses, secretion of IL-6 by dendritic cells (DCs) maintained autoreactive B cells in an unresponsive state. In this study, we describe that TLR4-activated DCs from lupus-prone mice are defective in repressing autoantibody secretion, coincident with diminished IL-6 secretion. Reduced secretion of IL-6 by MRL/lpr DCs reflected diminished synthesis and failure to sustain IL-6 mRNA production. This occurred coincident with lack of NF-kappaB and AP-1 DNA binding and failure to sustain IkappaBalpha phosphorylation. Analysis of individual mice showed that some animals partially repressed Ig secretion despite reduced levels of IL-6. This suggests that in addition to IL-6, DCs secrete other soluble factor(s) that regulate autoreactive B cells. Collectively, the data show that MRL/lpr mice are defective in DC/IL-6-mediated tolerance, but that some individuals maintain the ability to repress autoantibody secretion by an alternative mechanism.     

Failure of CD25+ T cells from lupus-prone mice to suppress lupus glomerulonephritis and sialoadenitis

The development of organ-specific autoimmune diseases in mice thymectomized on day 3 of life (d3tx mice) can be prevented by transferring CD4(+)CD25(+) T cells from syngeneic, normal adult mice. Using a d3tx model, we asked whether CD4(+)CD25(+) T cell deficiency contributes to glomerulonephritis (GN) in lupus-prone mice. New Zealand Mixed 2328 (NZM2328) mice spontaneously develop autoantibodies to dsDNA and female-dominant, fatal GN. After d3tx, both male and female NZM2328 mice developed 1) accelerated dsDNA autoantibody response, 2) early onset and severe proliferative GN with massive mesangial immune complexes, and 3) autoimmune disease of the thyroid, lacrimal gland, and salivary gland. The d3tx male mice also developed autoimmune prostatitis. The transfer of CD25(+) cells from 6-wk-old asymptomatic NZM2328 donors effectively suppressed dsDNA autoantibody and the development of autoimmune diseases, with the exception of proliferative lupus GN and sialoadenitis. This finding indicates that NZM2328 lupus mice have a selective deficiency in T cells that regulates the development of lupus GN and sialoadenitis. After d3tx, the proliferative GN of female mice progressed to fatal GN, but largely regressed in the male, thereby revealing a checkpoint in lupus GN progression that depends on gender.     

The effect of thymectomy on lupus-prone mice

The effect of neonatal thymectomy on the induction and/or modification of murine SLE disease was examined in several representative groups of mice with early-life SLE (MRL/Mp-lpr/lpr females, BXSB males, (NZB X W)F1 females, (NZW X BXSB)F1 males and females), late-life SLE (MRL/Mp-+/+ and BXSB females), and normal strains (BALB/c and C57BL/6 females). Our results indicated that thymectomy prevented disease only in the MRL/Mp-lpr/lpr SLE mice, and that this effect diminished as thymectomy was delayed beyond 3 wk post-natally. In the other SLE mice studied, neonatal thymectomy did not modify disease symptoms to any significant degree. Moreover, depletion of mature T cells from donor BXSB male bone marrow did not affect the expression of early-life SLE in thymectomized BXSB female recipients. Neonatal thymectomy did not induce SLE in normal mice. Of note, neonatal thymectomy did not completely deplete the Thy-1.2+ cell population, i.e., 10 to 15% remained in the spleens of the thymectomized mice. This incomplete T cell depletion, together with the previously demonstrated dependence on and hyperresponsiveness of BXSB and (NZB X W)F1 B cells to T helper cell-derived accessory signals, cast doubts on earlier conclusions that B cells from some SLE mice can autonomously proliferate and differentiate to autoantibody-secreting cells. It seems more appropriate to conclude that B cells from the various SLE mice vary in their degree of response to, and production of, T cell-derived helper signals, and thus in their expression of B cell hyperactivity and disease.     

Altered chemokine production and accumulation of regulatory T cells in intestinal adenomas of APCMin/+ mice

Tumor progression in the colon moves from aberrant crypt foci to adenomatous polyps to invasive carcinomas. The composition of the tumor-infiltrating leukocyte population affects the ability of the immune system to fight the tumor. T cell infiltration into colorectal adenocarcinomas, particularly T helper 1 (Th1) type T cells as well as increased regulatory T cell (Treg) frequencies, is correlated with improved prognosis. However, whether Th1 cells and Tregs are already present at the adenoma stage is not known. In this study, the APC^Min/+ mouse model of intestinal adenomatous polyposis was used to investigate tumor-associated lymphocyte subsets and the mechanisms of their accumulation into gastrointestinal adenomas. Compared to unaffected tissue, adenomas accumulated CD4⁺FoxP3⁺ putative Treg in parallel with lower frequencies of conventional T cells and B cells. The accumulation of Treg was also observed in human adenomatous polyps. Despite high Treg numbers, the function of conventional T cells present in the APC^Min/+ adenomas was not different from those in the unaffected tissue. Adenomas displayed an altered chemokine balance, with higher CCL17 and lower CXCL11 and CCL25 expression than in the unaffected tissue. In parallel, CXCR3⁺ Tregs were largely absent from adenomas. The data indicate that already in early stages of tumor development, the balance of lymphocyte-recruiting chemokines is altered possibly contributing to the observed shift toward higher frequencies of Treg.     

Microtubules of guard cells are light sensitive

Guard cells of stomata are characterized by ordered bundles of microtubules radiating from the ventral side toward the dorsal side of the cylindrical cell. It was suggested that microtubules play a role in directing the radial arrangement of the cellulose micro-fibrils of guard cells. However, the role of microtubules in daily cycles of opening and closing of stomata is not clear. The organization of microtubules in guard cells of Commelina communis leaves was studied by analysis of three-dimensional immunofluorescent images. It was found that while guard cell microtubules in the epidermis of leaves incubated in the light were organized in parallel, straight and dense bundles, in the dark they were less straight and oriented randomly near the stomatal pore. The effect of blue and red light on the organization of guard cell microtubules resembled the effects of white light and dark respectively. When stomata were induced to open in the dark with fusicoccin, microtubules remained in the dark configuration. Furthermore, when incubated in the light, guard cell microtubules were more resistant to oryzalin. Similarly, microtubules of Arabidopsis guard cells, expressing green fluorescent protein-tubulin alpha 6, were disorganized in the dark, but were organized in parallel arrays in the presence of white light. The dynamics of microtubule rearrangement upon transfer of intact leaves from dark to light was followed in single stomata, showing that an arrangement of microtubules typical for light conditions was obtained after 1 h in the light. Our data suggest that microtubule organization in guard cells is responsive to light signals.     

Transgenic mice expressing the diphtheria toxin receptor are sensitive to the toxin

Whereas diphtheria and the mechanism of action of diphtheria toxin, the bacterial molecule that induces the disease, have been studied and understood for some time, the receptor that allows animal cells to bind the toxin escaped identification until recently. The receptor was identified by its ability to confer toxin-sensitivity to mouse cells, which are normally toxin-resistant. Although mice are also naturally resistant, we now demonstrate that transgenic mice expressing the diphtheria toxin receptor are as sensitive to the toxin as are humans and other toxin-sensitive animals. These transgenic mice provide a suitable model for studying modern antidotes for diphtheria.     

Mast cell hyperplasia in the skin of Dsg4-deficient hypotrichosis mice, which are long-living mutants of lupus-prone mice

Desmosomal cadherins are essential cell adhesion molecules expressed in the epidermis. We identified a mutation of a cadherin superfamily member, namely, desmoglein 4 (Dsg4), in early onset of death (EOD)( hage ) mice with hypotrichosis. The mutation was induced by the insertion of an early transposon II-beta into intron 8 of Dsg4. Mast cell hyperplasia was observed in the skin of EOD( hage ) mice. The abnormally expanded population of lpr T cells, i.e., CD4(-)CD8(-)B220(+)Thy1.2(+) alphabetaT cells, in the splenocytes of EOD mice was reduced in EOD( hage ) mice. Therefore, it was suspected that the long-living mutant EOD( hage ) mice were selected from lupus-prone EOD mice because of their immunological immaturity. These findings clearly indicate that Dsg4 is an important molecule for the formation of hair follicles and hypothesize that unorganized hyperplastic hair follicles in anagen due to the Dsg4 mutation provide niches for mast cell precursors in the skin.     

Deficiency of P-selectin or P-selectin glycoprotein ligand-1 leads to accelerated development of glomerulonephritis and increased expression of CC chemokine ligand 2 in lupus-prone mice

The selectins and their ligands mediate leukocyte rolling on endothelial cells, the initial step in the emigration cascade leading to leukocyte infiltration of tissue. These adhesion molecules have been shown to be key promoters of acute leukocyte emigration events; however, their roles in the development of long-term inflammatory responses, including those that occur during chronic inflammatory diseases such as systemic lupus erythematosus, are unclear. To assess participation of P-selectin in such disorders, we studied the progression of systemic lupus erythematosus-like disease in P-selectin-deficient and control MRL/MpJ-Fas(lpr) (Fas(lpr)) mice. Surprisingly, we found that P-selectin deficiency resulted in significantly earlier mortality, characterized by a more rapid development of glomerulonephritis and dermatitis. Expression of CCL2 (MCP-1) was increased in the kidneys of P-selectin mutant mice and in supernatants of LPS-stimulated primary renal endothelial cell cultures from these mice. A closely similar phenotype, including elevated renal expression of CCL2, was also observed in Fas(lpr) mice deficient in the major P-selectin ligand, P-selectin glycoprotein ligand-1. These results indicate that P-selectin and P-selectin glycoprotein ligand-1 are not required for leukocyte infiltration and the development of autoimmune disease in Fas(lpr) mice, but rather expression of these adhesion molecules is important for modulating the progression of glomerulonephritis, possibly through down-regulation of endothelial CCL2 expression.     

Werner syndrome lymphoblastoid cells are sensitive to camptothecin-induced apoptosis in S-phase

Werner Syndrome (WRN) is an autosomal recessive disorder showing an endogenous mutator phenotype in combination with an elevated risk of predominantly mesenchymal cancer. The gene mutated in WRN patients codes for 3’→5’ DNA helicase and 3’→5’ exonuclease activities. We have found similar S-phase arrest in both WRN and control cells after treatment with the DNA-topoisomerase-I-trapping drug camptothecin; this may be responsible for the drug-exposure-related growth inhibition seen in both cell types. A clearer phenotypic difference between WRN and control immortalized B-cell lines (LCLs) is obtained by examining cell death. The mechanism of camptothecin-induced cell death in WRN-deficient LCLs appears to be through apoptosis, a phenotype that strongly differentiates WRN-deficient from wild-type LCLs. We hypothesize that, in cells deficient for WRN function, a topoisomerase-I-DNA intermediate persists. Conflict with DNA replication may lead to apoptosis, increased mutation rates, and cancer in WRN. Received: 23 September 1998 / Accepted: 26 October 1998     

Regulation of T cell-dependent autoantibody production by a gammadelta T cell line derived from lupus-prone mice

Lupus-prone (MRLxC57BL/6) F(1) mice lacking gammadelta T cells show more severe lupus than their T cell-intact counterparts, suggesting that gammadelta T cells down-modulate murine lupus. To determine the mechanisms for this effect, we assessed the capacity of gammadelta T cell lines derived from spleens of alphabeta T cell-deficient MRL/Mp-Fas(lpr) (MRL/Fas(lpr)) mice to down-regulate anti-dsDNA production generated by CD4(+)alphabeta T helper cell lines and activated B cells from wild-type MRL/Fas(lpr) mice. One line, GD12 (gd TCR(+), CD4(-)CD8(-)), had the capacity to reduce anti-dsDNA production in a contact-dependent manner. GD12 also killed activated MRL/Fas(lpr) (H-2(k)) B cells, with less cytolysis of resting B cells than that generated by in comparison to cytokine-matched gammadelta T cell lines. In addition, GD12 also killed activated B cells derived from C57BL/6-Fas(lpr) (H-2(b)) or beta(2)-microglobulin (beta(2) M)-deficient MRL/Fas(lpr) mice, suggesting cytolysis was neither MHC- nor CD1-restricted. Killing by GD12 was inhibited by anti-TNFalpha and anti-TNF-R1, and partially blocked by anti-gd TCR Fab fragments, but not by anti-FasL, anti-TNF-R2 (p75) or concanamycin A. IL-10 produced by GD12 also partially inhibited alphabeta Th1-dependent but not alphabeta Th2-dependent autoantibody production. These findings prove that we have identtified a gammadelta T cell line that suppresses autoantibody synthesis by alphabeta T-B cell collaboration in vitro.     

CD4+ T cells from lupus-prone mice avoid antigen-specific tolerance induction in vivo

Activated T cells in spontaneous lupus presumably bypass normal tolerance mechanisms in the periphery, since thymic tolerance appears intact. To determine whether such T cells indeed avoid in vivo peripheral tolerance mechanisms, we assessed their activation and recall responses after in vivo Ag stimulation in the absence of exogenously supplied costimulatory signals. Naive CD4(+) AND (transgenic mice bearing rearranged TCR specific for pigeon cytochrome c, peptides 88-104) TCR-transgenic T cells, specific for pigeon cytochrome c, from lupus-prone Fas-intact MRL/Mp+(Fas-lpr) and from H-2(k)-matched control CBA/CaJ and B10.BR mice (MRL.AND, CBA.AND, and B10.AND, respectively) were adoptively transferred into (MRL x CBA)F(1) or (MRL x B10)F(1) recipients transgenically expressing membrane-bound pigeon cytochrome c as a self-Ag. MRL.AND and control CBA.AND and B10.AND-transgenic T cells were activated and divided after transfer, indicating encounter with their cognate Ag; however, T cells from CBA.AND and B10.AND mice were impaired in their ability to proliferate and produce IL-2 after challenge with pigeon cytochrome c in ex vivo recall assays, a typical phenotype of anergized cells. By contrast, MRL.AND T cells proliferated more, and a significantly higher percentage of such cells produced IL-2, compared with control T cells. This observation that MRL T cells avoided anergy induction in vivo was confirmed in an in vitro system where the cells were stimulated with an anti-CD3 in the absence of a costimulatory signal. These experiments provide direct evidence that CD4(+) T cells from Fas-intact lupus-prone MRL mice are more resistant than nonautoimmune control cells to anergy induction. Anergy avoidance in the periphery might contribute to the characteristic finding in lupus of inappropriate T cell activation in response to ubiquitous self-Ags.     

Epstein-Barr virus-infected Akata cells are sensitive to histone deacetylase inhibitor TSA-provoked apoptosis

Epstein-Barr virus (EBV) infects more than 90 % of the world's population and has a potential oncogenic nature. A histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), has shown potential ability in cancer chemoprevention and treatment, but its effect on EBV-infected Akata cells has not been examined. This study investigated the effect of TSA on the proliferation and apoptosis of the cells. TSA inhibited cell growth and induced cytotoxicity in the EBV-infected Akata cells. TSA treatment sensitively induced apoptosis in the cell, which was demonstrated by the increased number of positively stained cells in the TUNEL assay, the migration of many cells to the sub-G0/G1 phase in flow cytometric analysis, and the ladder formation of genomic DNA. Western blot analysis showed that caspase-dependent pathways are involved in the TSA-induced apoptosis of EBV-infected Akata cells. Overall, this study shows that EBV-infected B lymphomas are quite sensitive to TSA-provoked apoptosis.     

Erythroid precursors cultured from adult mice are sensitive to H2O2 toxicity

Summary The growth of late erythroid precursors (CFU-Es) from adult bone marrow is inhibited when Iscove's modified Dulbecco's medium supplied in liquid form is used. Catalase and other H₂O₂ destroying compounds restore the capacity of culture medium to support colony development. However early precursors from adult bone marrow and fetal liver CFU-Es were resistant to H₂O₂. This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale and the Fédération Nationale des Centres de Lutte contre le Cancer.     

Epithelial cells are sensitive detectors of bacterial pore-forming toxins

Epithelial cells act as an interface between human mucosal surfaces and the surrounding environment. As a result, they are responsible for the initiation of local immune responses, which may be crucial for prevention of invasive infection. Here we show that epithelial cells detect the presence of bacterial pore-forming toxins (including pneumolysin from Streptococcus pneumoniae, alpha-hemolysin from Staphylococcus aureus, streptolysin O from Streptococcus pyogenes, and anthrolysin O from Bacillus anthracis) at nanomolar concentrations, far below those required to cause cytolysis. Phosphorylation of p38 MAPK appears to be a conserved response of epithelial cells to subcytolytic concentrations of bacterial poreforming toxins, and this activity is inhibited by the addition of high molecular weight osmolytes to the extracellular medium. By sensing osmotic stress caused by the insertion of a sublethal number of pores into their membranes, epithelial cells may act as an early warning system to commence an immune response, while the local density of toxin-producing bacteria remains low. Osmosensing may thus represent a novel innate immune response to a common bacterial virulence strategy.     

p53 checkpoint-defective cells are sensitive to X rays, but not hypoxia

X-ray-induced damage leads to cell-cycle "checkpoint" arrest by p53-dependent induction of the cyclin-dependent kinase inhibitor p21 (Waf1/Cip1/Sdi1). Human tumor cells that lack this response fail to arrest after exposure to DNA-damaging agents, undergo multiple rounds of endoreduplicative DNA synthesis, and eventually commit to an apoptotic cell death. Since low oxygen tension can also induce p53 protein accumulation, and can lead to cell-cycle arrest or apoptosis, we examined the expression of p21 in tumor cells under normoxic and hypoxic conditions. In a survey of cells, mRNA for the p21 gene was induced two- to threefold in response to hypoxia in a seemingly p53-independent manner. We therefore examined genetically matched cells that differ in their p21 and p53 status for response to ionizing radiation and hypoxia. We found that both p21-deficient and p53-deficient cells exhibit an increase in chromosome instability, an increased level of apoptosis, and a failure to arrest after exposure to ionizing radiation. However, cells that lack either p21 or p53 exhibit no increase in chromosome instability or elevated apoptosis and still arrest in response to hypoxia. Thus, the mechanism responsible for the differential response to either hypoxia or X rays presumably lies in the control of cell-cycle progression in response to stress and its dependence on p21. Since the loss of a DNA-damage-dependent checkpoint does not sensitize cells to killing by stresses that elicit a DNA-damage-independent checkpoint, targeting the function of p21 pharmacologically will not kill tumor cells in situ in the absence of a DNA damage signal.     

Drug-resistant breast carcinoma (MCF-7) cells are paradoxically sensitive to apoptosis

The purpose of this study was to determine whether expression of tissue transglutaminase (TG2) and caspase-3 proteins in drug-resistant breast carcinoma MCF-7/DOX cells would render these cells selectively susceptible to apoptotic stimuli. Despite high resistance to multidrug resistance (MDR)-related drug, doxorubicin (> or =150-fold), the MCF-7/DOX cells were extremely sensitive to apoptotic stimuli. Thus, calcium ionophore, A23187 (A23187) and the protein kinase C inhibitor staurosporine (STS) each induced rapid and time-dependent apoptosis in MCF-7/DOX cells. The apoptosis induced by either agent was accompanied by caspase-3 activation and other downstream changes that are typical of cells undergoing apoptosis. The alterations upstream of caspase-3 activation, however, such as loss in mitochondrial membrane potential (DeltaPsi), release of cytochrome c, and activation of caspase-8, and caspase-9, were detected only in STS-treated cells. The A12387 failed to induce any of the caspase-3 upstream changes, implying that A23187-induced apoptosis may utilize one or more novel upstream pathways leading to the activation of caspase 3. In summary, these data demonstrate that MCF-7/DOX cells are much more sensitive to apoptotic stimuli than previously thought and that A23187-induced apoptosis may involve some novel, yet unidentified, upstream pathway that leads to the activation of caspase-3 and other downstream events.