Age-associated modulation of apoptosis and activation in murine B lymphocytes

We examined neuronal activity in three motor cortical areas while a rhesus monkey adapted to novel visuomotor transforms. The monkey moved a joystick that controlled a cursor on a video screen. Each trial began with the joystick centered. Next, the cursor appeared in one of eight positions, arranged in a circle around a target stimulus at the center of the screen. To receive reinforcement, the monkey moved the joystick so that the cursor contacted the target continuously for Is. The video monitor provided continuous visual feedback of both cursor and target position. With those elements of the task constant, we modified the transform between joystick movement and that of the cursor at the beginning of a block of trials. Neuronal activity was studied as the monkey adapted to these novel joystick-cursor transforms. Some novel tasks included spatial transforms such as single-axis inversions, asymmetric double-axis inversions and angular deviations (also known as rotations). Other tasks involved changes in the spatiotemporal pattern and magnitude of joystick movement. As the monkey adapted to various visuomotor tasks, 209 task-related neurons (selected for stable background activity) showed significant changes in their task-related activity: 88 neurons in the primary motor cortex (M1), 32 in the supplementary motor cortex (M2), and 89 in the caudal part of the dorsal premotor cortex (PMdc). Slightly more than half of the sample in each area showed significant changes in the magnitude of activity modulation during adaptation, with the number of increases approximately equaling the number of decreases. These data support the prediction that changes in task-related neuronal activity can be observed in M1 during motor adaptation, but fail to support the hypothesis that M1 and PMdc differ in this regard. When viewed in population averages, motor cortex continued to change its activity for at least dozens of trials after performance reached a plateau. This slow, apparently continuing change in the pattern and magnitude of task-related activity may reflect the initial phases of consolidating the motor memory for preparing and executing visuomotor skills.     

In vivo expression of interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, tumour necrosis factor-alpha and interferon-gamma in the fetal murine thymus

Cytokines are known to play a role in T-cell lymphopoiesis as potent growth or differentiation factors, but many experiments focusing on their role in the thymus have been conducted only in vitro. We have thus used frozen sections obtained from fetal thymuses of normal C57BL 6 mice to investigate by immunohistochemistry the presence of interleukin-1 beta (I4-1 beta), IL-2. IL-4. IL-6. interferon-7 (IFN-7) and tumour necrosis facor-alpha (TNF-alpha). The results reveal that apart from IL-2, which was not detected, all these cytokines display a time-dependent expression pattern in the normal fetal thymus. First, production of IL-4, IL-6 and TNF-alpha is detected around days 13 14; this is followed by a second wave on days 16 17, with a production of IL-1 beta, IL-4 and IL-6, and finally, just before birth (day 19), by a third wave of IL-1 beta, IL-4, IL-6, IFN-7 and TNF-alpha production. This supports the hypothesis that cytokines play a rote in T-cell lymphopoiesis.     

Interleukin-6 (IL-6) inhibits thyroid function in the presence of soluble IL-6 receptor in cultured human thyroid follicles

Interleukin-6 (IL-6), a pleiotropic cytokine, is postulated to be involved in the pathogenesis of sick euthyroid syndrome, although the direct in vitro effects of IL-6 on human thyroid function are controversial. Because IL-6 signal can be transduced when the complex of IL-6 and soluble IL-6 receptor (sIL-6R) binds to gp 130, an IL-6 signal transducer, we studied the effects of IL-6 and sIL-6R on thyroid function, using human thyroid follicles obtained from patients with Graves' disease. IL-6 alone had no inhibitory effect on TSH-induced thyroid function (125I incorporation and organic 125I release), even at supraphysiological concentrations. However, in the presence of physiological concentrations of sIL-6R (100 ng/ml), IL-6 inhibited thyroid function dose dependently and completely, accompanied with the decreased ratio of 125I-T3/125I-T4 not only in the thyroid follicles but also in the culture medium. Thyroid follicles did not secrete sIL-6R but produced IL-6 constitutively. Consistent with these findings, sIL-6R inhibited thyroid function slightly at high concentrations. Furthermore, RT-PCR analyses revealed that human thyroid follicles expressed the messenger RNAs for IL-6 and gp130 but scarcely messenger RNA for IL-6R. These in vitro findings suggest that IL-6 alone hardly affects thyroid function in thyroid follicles in which IL-6R gene is scarcely expressed. However, because sIL-6R is present abundantly in serum, IL-6 in vivo would be capable of inhibiting the synthesis and release of T4 and, to a greater extent, T3 from the thyroid gland. These in vitro findings are at least partly related to the development of sick euthyroid syndrome.     

Characterization of the antitumor immune response generated by treatment of murine tumors with recombinant adenoviruses expressing HSVtk, IL-2, IL-6 or B7-1

In a cancer gene therapy model recombinant adenoviruses expressing the herpes simplex virus thymidine kinase (HSVtk) gene were injected into tumors in situ, either alone or in combination with adenoviruses (Avs) engineered to express IL-2, IL-6 or the costimulatory molecule B7-1. HSVtk phosphorylates the prodrug ganciclovir, thus converting it into an antimetabolite which kills not only HSVtk expressing cells, but also by the 'bystander effect', neighboring untransduced tumor cells. The tumors regressed in 80% of mice upon AvTK/ganciclovir treatment: combinations with AvIL-2, AvIL-6, or AvB7-1 did not improve these results. Cured mice were protected from further challenge with wild-type tumor but not from challenges with an unrelated syngeneic tumor cell line. Since cytotoxic T lymphocyte responses in this tumor model were weak, we analyzed cytokine secretion from spleen cells of treated animals. The best correlate of antitumor immunity in this model was enhanced secretion of GM-CSF, while secretion of IL-2, IL-6 and IFN gamma was also frequently increased but not as consistently. The enhanced IFN gamma secretion associated with unchanged IL-4 secretion suggests that AvTK treatment results in a predominantly Th1-mediated antitumor immune response.     

Characterization of the mitogenic response of murine CD5+ and conventional B lymphocytes to lipopolysaccharide

The nature of the response of conventional and CD5+ B cells to stimulation in vitro with optimal mitogenic concentrations of LPS was examined to elucidate the contributions of these B cell subsets in polyclonal B lymphocyte responses. Stimulation of murine splenic lymphocytes with LPS resulted in an increase in total biomass, peaking at 72 h of culture. The viability of the cultures remained high (> 90%) until 48 h of culture. A combination of trypan blue and 7-aminoactinomycin D (7AAD) exclusion in conjunction with PE-anti-CD5 and FITC-anti-B220 enabled more detailed analysis of the cultures. The total number of conventional B cells, viable and non-viable, increased until 48 h of culture and then decreased when stimulated with LPS, while CD5+ B cells increased over the culture period. The numbers of conventional B cells in the control cultures decreased, but the CD5+ B cell numbers remained stable. An examination of the modes of death of the B cell subsets using 7AAD showed that unstimulated conventional B cells were apoptotic rather than degenerate but, following stimulation with LPS, apoptotic and degenerate cells were found. Apoptotic and degenerate CD5+ B cells were found in both stimulated and unstimulated cultures, but the percentage of these apoptotic and degenerate cells was increased significantly only at 72 h and 96 h of culture in stimulated cultures compared with 24 h onwards in the control cultures. Morphological analysis and gel electrophoretic studies of extracted DNA reflected these findings. It was also found that the increase in the number and percentage of non-viable cells in the cultures was not equal to the decrease in the number and percentage of viable cells. Activation of B cells was examined using expression of B7-1 (CD80) as a marker. When stimulated with LPS a greater proportion of conventional B cells expressed B7-1 after 24 h of culture than in the control cultures; however, only at 72 h and 96 h of culture was the proportion of CD5+ B cells expressing B7-1 significantly higher than in the control cultures. These results show that conventional B cells are stimulated to proliferate and to become activated by LPS and that death is apoptotic rather than degenerate or necrotic. CD5+ B cells were also shown to be stimulated by LPS; they became activated and death was delayed. The data suggest that in addition to the proliferative role, LPS acts to delay death and to activate conventional and CD5+ B cells.     

Extracellular 37-kDa antigenic protein from Actinobacillus actinomycetemcomitans induces TNF-alpha, IL-1 beta, and IL-6 in murine macrophages

The extracellular antigens of Actinobacillus actinomycetemcomitans Y4 (serotype b) contain a 37-kDa protein which is a major target for IgGs from patients suffering from severe alveolar bone loss. Since the 37-kDa protein has not been studied sufficiently, our investigation focused on its characteristics, e.g., its localization, specificity, and whether it directly stimulates macrophages to produce cytokines. The 37-kDa protein was purified from the culture supernatant of the Y4 strain by means of chromatofocusing and gel filtration. The 37-kDa protein is a unique glycoprotein which forms immune complexes with monoclonal antibodies against rhamnose-fucose polysaccharide. Patients with A. actinomycetemcomitans-associated periodontitis had higher antibody titers to the purified 37-kDa protein than healthy subjects (p < 0.001). Anti-37-kDa protein antibodies recognized a 37-kDa band in the cytosolic, ribosomal, and total membrane fractions from Y4 cells. Extracellular substances from other strains of A. actinomycetemcomitans (serotypes a and c) also reacted in the Western blots, but Haemophilus spp. or several periodontopathic bacteria did not. These results suggested that the 37-kDa protein is a cytosolic protein that is passed through the cell membrane, and its protein portion is specific for A. actinomycetemcomitans but common to serotypes. This protein induced Il-1 beta, Il-6, and TNF-alpha release from murine macrophages. The Il-6-inducing activity of the 37-kDa protein was higher than that of LPS. These findings suggested that the 37-kDa protein which is released from live cells plays a role in A. actinomycetemcomitans-associated periodontitis, as antigen inducing the release of inflammatory cytokines which are associated with alveolar bone loss.     

Role of IL-6 and the soluble IL-6 receptor in inhibition of VCAM-1 gene expression

Adhesion molecules such as VCAM-1 and ICAM-1 are increased in the central nervous system (CNS) during inflammatory responses and contribute to extravasation of leukocytes across the blood-brain barrier (BBB) and into CNS parenchyma. Astrocytes contribute to the structural integrity of the BBB and can be induced to express VCAM-1 and ICAM-1 in response to cytokines such as TNF-alpha, IL-1beta, and IFN-gamma. In this study, we investigated the influence of IL-6 on astroglial adhesion molecule expression. IL-6, the soluble form of the IL-6R (sIL-6R), or both IL-6 plus sIL-6R, had no effect on VCAM-1 or ICAM-1 gene expression. Interestingly, the IL-6/sIL-6R complex inhibited TNF-alpha-induced VCAM-1 gene expression but did not affect TNF-alpha-induced ICAM-1 expression. The inhibitory effect of IL-6/sIL-6R complex was reversed by the inclusion of anti-IL-6R and gp130 Abs, demonstrating the specificity of the response. A highly active fusion protein of sIL-6R and IL-6, covalently linked by a flexible peptide, which is designated H-IL-6, also inhibited TNF-alpha-induced VCAM-1 expression. sIL-6R alone was an effective inhibitor of TNF-alpha-induced VCAM-1 due to endogenous IL-6 production. These results indicate that the IL-6 system has an unexpected negative effect on adhesion molecule expression in glial cells and may function as an immunosuppressive cytokine within the CNS.     

The control of IL-4 gene expression in activated murine T lymphocytes: a novel role for neu-1 sialidase

IL-4 is important in controlling the development of immune responses. Following activation with anti-CD3epsilon under serum-free conditions, splenocytes from most normal (neu-1b) mouse strains directly produced IL-4 and other T cell cytokines. However, splenic T cells from SM/J and B10.SM (H-2v, neu-1a) strain mice, deficient in neu-1 sialidase activity, failed to produce IL-4 but produced normal levels of IL-2 following activation. Moreover, sialidase-deficient mice produced markedly less IgE and IgG1 Abs following immunization with protein Ags than did mouse strains with normal neu-1 sialidase activity. Enriched T cells from neu-1a mice failed to be effectively primed with exogenous murine IL-4 to become IL-4-producing cells. Treatment of splenocytes or enriched T cells from neu-1a mice with bacterial sialidase prior to activation or IL-4 priming promoted their subsequent capacity to produce IL-4. In contrast, activation of T cells from neu-1b mice in the presence of a sialidase inhibitor almost completely blocked subsequent IL-4 production. The presence of IL-4 during priming enhanced T cell expression of neu-1-specific sialidase activity and increased the membrane expression of asialo-G(M1) compared with T cells activated without IL-4. These results suggest that T cell-associated neu-1 sialidase is required for early IL-4 production by splenic T cells and is involved in the IL-4 priming process of conventional T cells to become active IL-4 producers.     

The influence of DNA structure on the in vitro stimulation of murine lymphocytes by natural and synthetic polynucleotide antigens

Although DNA is generally considered to be a poor immunogen, recent evidence suggests that DNA from various species differ in their immunological activity and that bacterial DNA can induce the in vitro proliferation of normal murine B cells. To delineate structural features of DNA associated with mitogenic activity, the response of murine lymphocytes to various natural and synthetic polynucleotides was determined. Both ss and dsDNA from two different bacterial strains were equally effective in inducing proliferation. This response was independent of adenosine methylation, since DNA from dam- Escherichia coli stimulated proliferation. Among the synthetic polymers tested, only the duplexes poly(dG).poly(dC), and poly(dG.dC) were mitogenic, while polymers containing dA, dT, or dI alone or in combination with dG and dC were inactive. The mitogenic activity of poly(dG.dC) was eliminated, however, upon substitution of rG for dG or 5medC for dC. The mitogenic activity did not require high molecular weight DNA since active polymers ranged in size from approximately 260 to 800 base pairs. In addition, E. coli DNA fragments of 50-300 and 125-600 bases were mitogenic. Together, these data suggest that the mitogenic activity of DNA is dependent on sequence-specific determinants that can be presented by synthetic DNA duplexes as well as bacterial ss and dsDNA.     

The cell-mediated response to schistosomal antigens at the clonal level. III. Identification of soluble egg antigens recognized by cloned specific granulomagenic murine CD4+ Th1-type lymphocytes

Granulomatous inflammation in schistosomiasis is a consequence of T cell-mediated hypersensitivity to parasite egg Ag. In the present study we used three consecutive independent chromatographic procedures to fractionate and identify the soluble egg Ag recognized by schistosome-specific, cloned, murine, CD4+ Th1-type lymphocytes, which had been shown previously to be capable of mediating granuloma formation in vivo when adoptively transferred to normal syngeneic hosts challenged with an i.v. injection of eggs. The stimulatory activity resided in two acidic egg molecules, with apparent molecular masses of 64 to 68 kDa and 38 to 42 kDa, each of which ran as a single band on SDS-PAGE after purification. Fast performance liquid chromatography and SDS-PAGE performed under reducing conditions suggested that the two molecules are related and that the 38- to 42-kDa molecule is a subunit of the 64- to 68-kDa molecule. Polyclonal lymphoid cells from schistosome-infected mice were similarly stimulated by the purified 64- to 68-kDa and 38- to 42-kDa molecules, implying that these are sensitizing Ag in the natural disease.     

Preparations of antigens and immunoadsorbents corresponding to the Streptococcus group A cell-wall polysaccharide

The allyl glycosides of a tri-, penta- and hexasaccharide corresponding to the Streptococcus Group A cell-wall polysaccharide were coupled to solid or soluble supports to give immunoaffinity columns and neoglycoproteins, respectively. Cysteamine hydrochloride was added to the allyl glycosides and the resulting cysteamine adducts were used for subsequent coupling to linkers via the amine functionality. The tri- and penta-saccharide cysteamine adducts were coupled directly to the azalactone-derivatized 3M Emphase Biosupport Medium AB 1 to yield two affinity columns. The penta- and hexa- saccharides were coupled to bovine serum albumin or ovalbumin via the conjugate addition of the epsilon-amino groups of lysines on the proteins with the N-acryloylated sugars or the oligosaccharide-squarate adducts, derived in turn from the cysteamine adducts. The efficiency of the above methods is compared.     

Molecular cloning of the murine IL-1 beta converting enzyme cDNA

IL-1 beta is a potent modulator of immune and inflammatory responses. Murine IL-1 beta is initially synthesized as an inactive 33-kDa pro-molecule that is activated by proteolytic cleavage between Asp-117 and Val-118 to generate the 17-kDa mature IL-1 beta protein. This cleavage is catalyzed by a specific protease that has been designated the IL-1 beta converting enzyme (or IL-1 beta convertase). We have used a human IL-1 beta convertase cDNA to isolate murine convertase cDNA from a WEHI-3 library. These cDNA predicted that the murine convertase is a 402-residue protein. Overall, the murine convertase showed 71% nucleotide and 62% predicted amino acid sequence identity with the human convertase. Southern blot analysis of interspecific backcross mice indicated that the murine IL-1 beta convertase is encoded by a single copy gene located on murine chromosome 9. The murine convertase showed broad constitutive expression, being detected in mononuclear phagocyte and T lymphocyte cell lines as well as in spleen, heart, brain, and adrenal glands. The expression of the murine convertase in mononuclear phagocytes was up-regulated by treatment with LPS or rIFN-gamma. These studies establish that the IL-1 beta convertase is an evolutionarily conserved, widely expressed enzyme that can be regulated at a pretranslational level.     

Critical role of IL-12 in dendritic cell-induced differentiation of naive B lymphocytes

Dendritic cells (DC) are potent APCs initiating immune responses. In a previous report, we demonstrated that DC directly enhance both proliferation and differentiation of CD40-activated naive and memory B cells. The present study deciphers the molecular mechanisms involved in DC-dependent regulation of B cell responses. Herein, we have identified IL-12 as the mandatory molecule secreted by CD40-activated DC that promote the differentiation of naive B cells into plasma cells secreting high levels of IgM. In fact, IL-12 synergizes with soluble IL-6R alpha-chain (sgp80), produced by DC, to drive naive B cell differentiation. IL-12 is critical for the differentiation of naive B cells into IgM plasma cells, whereas IL-6R signaling mainly promotes Ig secretion by already differentiated B cells. The differentiation of naive B cells in cocultures of B cells, T cells, and DC is IL-12 dependent, definitely demonstrating that the role of DC in humoral responses is not confined to the activation of T cells and further extending the physiologic relevance of DC/B cell interaction. Finally, this study also identifies differential requirements for DC-dependent naive and memory B cell differentiation, the latter being IL-12 independent. Altogether these results indicate that, in addition to prime T cells toward Thl development, DC, through the production of IL-12, may also directly signal naive B cell during the initiation of the immune response.     

IL-10-driven immunoglobulin production by B lymphocytes from IgA-deficient individuals correlates to infection proneness

In search for a possible explanation of the phenotypic heterogeneity in IgA deficiency, we studied the function of B cells from IgA-deficient (IgAd) individuals. Two groups of IgAd individuals, one frequently infected and one clinically apparently healthy, as well as normal controls, were studied. Peripheral blood mononuclear cells (PBMC) and B cells from IgAd individuals and controls were cultured with Staphylococcus aureus Cowan I strain and with anti-CD40 MoAb presented on the CD32-transfected fibroblast cell line in the presence of IL-10. In this experimental system PBMC and B cells from the infection-prone IgAd individuals produced only minute amounts of IgA. In contrast, PBMC and B cells from healthy IgAd subjects secreted significantly more IgA1 and IgA2 in comparison with infection-prone IgAd patients (P < 0.05). These data suggest that the abnormalities of B cell differentiation in IgAd could be of heterogeneous origin. Thus, whereas in healthy IgAd subjects IgA production may be efficiently up-regulated in vitro by addition of IL-10 to CD40-activated B cell culture, the corresponding B cell differentiation does not occur in infection-prone IgAd patients. These observations provide a conceptual framework for phenotypic heterogeneity in IgAd subjects.     

The granulomatous response in murine Schistosomiasis mansoni does not switch to Th1 in IL-4-deficient C57BL/6 mice

IL-4 plays an important role in polarizing inflammation toward a Th2 response. It remains uncertain, however, whether IL-4 also serves to prevent expression of Th1 inflammation. Therefore, using a genetically pure C57BL/6 IL-4-deficient mouse, we studied the role of IL-4 in regulating the production of IFN-gamma and Th1 inflammation in the granulomas of mice infected with Schistosoma mansoni. In contrast to normal animals, IL-4 mutant mice generated smaller liver granulomas that contained fewer eosinophils and no mast cells. Collagenase-dispersed granuloma cells were analyzed by flow cytometry and cultured in vitro to measure cytokine and Ig production. Compared with control granuloma cells, IL-4-/- cells secreted only small quantities of IL-5 and IL-10. Also, there was impaired expression of the IL-4-dependent molecules IgE and IgG1 as well as B cell surface class II and CD23. Yet the granulomas of IL-4 -/- animals produced little IFN-gamma, IgG2a, or other molecules associated with Th1 inflammation even after Ag or anti-CD3 stimulation. Splenocytes from IL-4 -/- animals stimulated with schistosome Ag also failed to produce a Th1 response. Our data show that most aspects of the Th2 response in murine schistosomiasis are highly dependent on IL-4 production. But in the absence of IL-4, neither the natural local granulomatous response to schistosome ova nor the systemic response to soluble egg Ag switches to the type 1 phenotype. Thus the production of IL-4 early in the inflammatory response is not the only factor preventing Th1 expression in inflammation.     

Antibody response of murine B1 cells to hen eggwhite lysozyme

Cell transfer studies were performed to investigate the ability of murine peritoneal B1 cells to produce specific IgG antibody against the T-dependent protein antigen, hen eggwhite lysozyme (HEL). Peritoneal cells (PeC) from normal BALB/c mice were transferred into newborn, allotype-congenic, C.B-17 severe combined immunodeficient (SCID) mice alone or together with splenic T cells from HEL-primed C.B-17 mice. After immunization with HEL, only those mice that received both PeC and primed T cells produced HEL-specific IgG of the PeC donor allotype. To identify the B cell subset responsible for antibody production, PeC were sorted before transfer into B1 and conventional B (B2) cell populations. It was found that transfer of purified B1 cells plus primed T cells resulted in high levels of IgG1 anti-HEL in approximately half of the SCID recipients, while mice receiving B2 cells produced little detectable antibody. The responses consisted primarily of IgG1 kappa anti-HEL, with no significant IgM or lambda-bearing components. Seventeen HEL-specific hybridomas of BALB/c origin, i.e., derived from the B1 cell donor, were obtained from reconstituted SCID mice after various periods of immunization and analyzed for fine specificity using a panel of avian lysozymes. All but one of the B1 cell-derived mAbs recognized an HEL epitope not present on the closely related bobwhite quail lysozyme (differing from HEL at only 4 of 129 amino acid positions). While IEF analyses demonstrated the presence of extensive clonotypic diversity, the epitope specificity pattern, which is rare among B2-cell-derived antibodies, suggests that the B1 cell recognition repertoire for HEL is severely limited.     

Specific sensitization of lymphocytes to tumor antigens by co-cultivation with peritoneal cells exposed to such antigens

Twenty-seven cases of congenital posterolateral diaphragmatic hernia past infancy are reviewed in tabular form and discussed as to presenting symptoms, physical and radiographic findings, operative treatment, and final outcome. A ten year old male treated by us is presented as a detailed case report. A great contrast is noted between the acute respiratory symptoms which threaten life in the infant hernia compared with the more chronic and recurrent gastrointestinal and respiratory symptoms in pateints past infancy. Onset of symptoms did not correlate with age or sex. Chest x-ray films and gastrointestinal contrast studies were most helpful in diagnosis. Abdominal and thoracic approaches were equally effective in reducing the herniated viscera and closing the diaphragmatic defect. We believe that long-term survival of patients with congenital posterolateral diaphragmatic hernia may be due to persistence of a confining pleuroperitoneal sac. Rupture of this sac in later life may coincide with onset of the characteristic symptoms which in turn prompt diagnostic studies. Congenital diaphragmatic hernia must be considered in the differential diagnosis of patients with both recurrent gastrointestinal and respiratory complaints. Plain radiographs of the chest and contrast studies of the gastrointestinal tract are necessary to confirm diagnosis preoperatively.     

Effects of vitamin B6 deficiency on cytokine levels and lymphocytes in mice

The effects of vitamin B6 (B6) deficiency on cytokine levels and proportions of lymphocyte subsets in BALB/c mice were investigated. The proportion of lymphocytes from the thymus and spleen of mice given no B6, that were CD4+ CD8- T cells, was larger than in mice given B6, and the ratio of CD8+ to CD4+ T cells in the thymus of mice given no B6 was lower. The concentrations of interleukin-5 and -10 in spleen cells stimulated in vitro with concanavalin A were significantly higher in the mice with B6 deficiency, as was their plasma corticosterone concentrations. These results suggested that B6 is necessary to maintain cytokine levels and lymphoid function in the thymus and spleen of mice.