Treatment of murine CD5- B cells with anti-Ig, but not LPS, induces surface CD5: two B-cell activation pathways.

Anti-Ig stimulated murine B cells express high levels of surface CD5 (ly-1) and increased CD44 while maintaining surface IgD, CD23 and J11d. Sorting of CD5- and CD5+ cells demonstrates that anti-Ig induces CD5 expression rather than the selective expansion of CD5+ cells. Anti Ig plus interleukin-6 (IL-6) induces the CD23, IgD, low ly-5 (B220) (CD45low), J11dhigh phenotype of typical CD5+ peritoneal B cells. In contrast, lipopolysaccharide (LPS)-stimulated B cells have high levels of CD44 but decreased surface IgD, CD23 and J11d and no CD5. Thus LPS and anti-Ig generate activated cells with differing phenotypes. Induced CD5+ cells have increased viability, even in the absence of added exogenous factors, while the viability of CD5- B cells is dependent on factors such as IL-4. We conclude that conventional CD5- B cells can be activated by either of two pathways: one generating CD5+ B cells; the other yielding conventional activated cells. We hypothesize that the first path requires slg cross-linking and corresponds to T-independent (type 2) stimulation, while cognate interaction with helper T cells in the absence of slg cross-linking induces B cells to enter the second path.     

Increased expression of intracellular HLA-DM but not on the surface of blood monocyte-derived dendritic cells during maturation

Cutaneous dendritic cells (DCs), Langerhans cells (LCs) and dermal dendritic cells (DDCs), are present in an immature state. The maturation of DCs is crucial for initiating an immune response. Since HLA-DM has an important role for antigen presentation, an increase in HLA-DM expression according to the maturation of blood monocyte-derived dendritic cells (MoDCs), which have similar characteristics with DDCs, is expected. Therefore, the aim of this study was to determine whether or not HLA-DM expression in MoDCs is related to maturation at each culture day (from day 0 to day 13) by flow cytometry. This was compared with the functional changes related to the maturation of MoDCs. MoDCs were generated by culturing human peripheral blood monocytes in the presence of GM-CSF and IL-4 for 7 days, which were followed by subsequent treatment with a cytokine cocktail (GM-CSF, IL-4, IL-1beta, TNF-alpha, IL-6 and PGE2) for the maturation of MoDCs. The intracellular HLA-DM was expressed in the immature MoDC. A sudden 3 to 8 fold increase in the intracellular HLA-DM expression was observed after treatment with a cytokine cocktail. HLA-DM was weakly expressed on the surface of the immature MoDC, but it seemed to be decreased with maturation. This study indicated that the intracellular HLA-DM expression increased, but not on the MoDC surface during maturation. This was despite the fact that HLA-DM expression was noted not only on the surface but also in the intracellular in the MoDC.     

Macrophage colony-stimulating factor gene transfer into tumor cells induces macrophage infiltration but not tumor suppression

In order to analyze the effect of a high local concentration of macrophage colony-stimulating factor (M-CSF; CSF-1) on tumor growth, the plasmacytoma cell line J558L was transfected with the human M-CSF gene and injected into syngeneic BALB/c mice. In contrast to the parental tumors, M-CSF transfectants were heavily infiltrated by macrophages as evidenced by immunohistochemistry with antibodies to Mac-1 and Mac-3 and by isolation of the macrophages from the tumor. Nevertheless, tumor growth was only slightly affected by M-CSF and M-CSF-producing cells grew as tumor in all cases. The growth retardation of M-CSF-producing cells varied depending on the experiment and seemed to be due to an indirect effect because the growth rate of the cells in vitro had not changed upon gene transfer. Attempts to activate the tumor-infiltrating macrophages for tumor suppression by systemic application of interferon-γ and/or lipopolysaccharide were not successful. Altogether, our results suggest that M-CSF is a potent chemoattractant for macrophages in vivo but alone is not sufficient to activate these macrophages for tumoricidal activity.     

Affinity maturation of B cells involves not only a few but a whole spectrum of relevant mutations

Affinity maturation of B lymphocytes within germinal centers involves both diversification of their B-cell receptors (BCRs) by somatic hypermutation (SHM) and a crucial receptor-mediated selection step. However, in contrast to recent advances in revealing the molecular mechanism of SHM, the fundamentals of the selection process are still poorly understood, i.e. it is often not clear how and how many mutations contribute to improving a BCR during the response against a given antigen. A general drawback in assessing the mutations relevant to the selection process is the difficult task of rating the relative contributions of selection and intrinsic biases to the experimentally observed mutation patterns of BCRs. The approach proposed here is premised on statistical comparison of the frequency distributions of nucleotide substitutions as observed in datasets of hypermutated BCRs against their frequency distribution expected under the null hypothesis of no selection. Thereby, we show that the spectrum of mutations relevant to maturation of canonical anti-(4-hydroxy-3-nitrophenyl)acetyl BCRs is much broader than previously acknowledged, going beyond the scope of single key mutations. Moreover, our results suggest that maturation not only involves selection by means of affinity but likewise expression and stabilization of BCRs.     

OmpA targets dendritic cells, induces their maturation and delivers antigen into the MHC class I presentation pathway

We analyzed the interaction between a bacterial cell wall protein and dendritic cells (DCs). Outer membrane protein A from Klebsiella pneumoniae (kpOmpA) specifically bound to professional antigen presenting cells and was endocytosed by immature DCs via a receptor-dependent mechanism. kpOmpA signaled through Toll-like receptor 2, induced DCs to produce interleukin 12 and induced maturation of DCs. Whole antigen that was coupled to kpOmpA and injected into mice was taken up by DCs and delivered to the conventional cytosolic MHC class I presentation pathway. kpOmpA also primed antigen-specific CD8+ CTLs in the absence of CD4+ T cell help or adjuvant and elicited therapeutic immunity to antigen-expressing tumors. Thus, OmpA belongs to a class of proteins that are able to elicit CTL responses to exogenous antigen.     

MRC OX19 recognizes the rat CD5 surface glycoprotein,but does not provide evidence for a population of CD5bright B cells

To clone the rat CD5 gene we first produced two rat CD5 probes. The probes were obtained by polymerase chain reaction (PCR) on rat genomic DNA using primers designed on conserved regions between mouse and human CD5. The screening of a rat cDNA library at high stringency using these probes resulted in a 1.5-kb positive clone. The DNA sequence of this clone confirmed its CD5 nature, but the clone appeared to lack part of the 5′ and part of the 3′ end. These missing 5′ and 3′ ends were obtained by PCR on rat thymus RNA. By ligating these PCR products to the original 1.5-kb CDM8 clone, a full-length rat CD5 gene was constructed. The full-length clone showed high identity with mouse and human CD5; however, at the 5′ site of the gene a region of 36 nucleotides is present which is not seen in either mouse or human CD5. We have evidence that this sequence is a normal constituent of the rat CD5 gene: first, it is in frame with the rest of the CD5 coding sequence; second, it does not contain a stop codon; and third, it is also present in the CD5 gene of other rat strains. We transfected the full-length CD5 construct in COS cells and demonstrated that indeed the CD5 protein is recognized by MRC OX19. Although we showed that CD5 mRNA is present in rat B cells, extensive flow cytometry analysis using MRC OX19 as antibody failed to detect B cells expressing significant levels of CD5 on their cell surface compared to other B cells in any tissue or cell suspension tested from a variety of rat strains. This is in contrast with the mouse where a distinct population of B cells (B-la cells) can be found expressing more CD5 than the other B cells. Either B-1 cells are not present in rats or CD5 is not the right phenotypic marker for rat B-1 cells. It still remains to be investigated whether a population of B cells with functions similar to those of murine B-1 cells is present in rats.     

Gamma interferon, but not Mycobacterium leprae, induces major histocompatibility class II antigens on cultured rat Schwann cells

Summary In order to investigate the possible role of Schwann cells in immune reactions, and in particular their involvement in the response to infection withMycobacterium leprae, it was determined under what conditions Schwann cells express major histocompatibility complex class II (MHC class II) antigens, since these molecules are thought to have a key role in antigen presentation during cellular immune responses. In situ andin vitro preparations from newborn and adult rat sciatic nerves were used as a model system to examine this question. Schwann cells in dissociated cell cultures did not express immunohistochemically detectable amounts of MHC class II antigens. Teased nerve preparations from the sciatic nerves of healthy adult rats showed no detectable immunolabelling of either myelin-forming or non-myelin-forming Schwann cells.When dissociated Schwann cell cultures derived from the sciatic nerves of either neonatal or adult rats were treated with 10, 50 or 100 units of gamma Interferon, MHC class II antigens were detectable on the surface of some Schwann cells 48 h after addition of the interferon. By 72 h, 32.29 ± 3.9% of Schwann cells in the cultures from neonatal rats and 53.32 ± 5.4% of Schwann cells in cultures from adult rats, identified by the presence of intracellular S-100, were clearly MHC class II-positive, especially at doses of 50 and 100 units per ml of gamma interferon. Some, but not all, of the fibroblastic cells were very weakly MHC class Il-positive. Infection of the cultures withMycobacterium leprae did not induce MHC class II antigen expression in either Schwann cells or fibroblasts.These results suggest that one of the functional roles of Schwann cells' is the presentation of foreign antigens to T lymphocytes during nerve infection, leading to activation or augmentation of the cellular immune response. With respect toMycobacterium leprae in particular, it is therefore possible that infected Schwann cells might be capable of participating in the normal immune response toMycobacterium leprae.     

Human C5a induces a substantial histamine release in human basophils but not in tissue mast cells

The histamine-releasing effect of human C5a was compared with that of concanavalin A in blood basophils and isolated adenoidal mast cells from the same donors. In basophils, C5a (10 ng/ml) induced a significant histamine release (7.5 +/- 2.1%, corrected value). In isolated adenoidal mast cells C5a had only a marginal effect (2% histamine release), although the cells responded markedly to concanavalin A stimulation (about 13% release). The results support the view that the heterogeneity of mast cells and basophils is also reflected in the expression of anaphylatoxin receptors on their surface.     

Dendritic cell maturation is induced by mycoplasma infection but not by necrotic cells

To identify environmental stimuli that induce dendritic cell (DC) maturation, we exposed human monocyte-derived immature DC to apoptotic or necrotic cells and measured the levels of expression of costimulatory molecules and cytokine production. While most necrotic or apoptotic cells did not have any effect, some induced DC maturation as detected by up-regulation of CD83 and B7.2 and production of IL-12 and IL-6. The capacity of these cell lines to induce DC maturation was due to their contamination by mycoplasma, since the maturation-inducing effect disappeared when the cells were treated with cyproxin. Furthermore, cell lines deliberately infected with mycoplasma containing supernatant acquired the capacity to induce DC maturation. Our results reveal that DC are able to sense mycoplasma infection and mature as they do in response to most viruses and bacteria. In contrast, apoptotic or necrotic cells fail to induce DC maturation.     

Naive CD8(+) but not CD4(+) T cells induce maturation of dendritic cells

We have shown previously that the generation of tumor-reactive CD8(+) cytotoxic T lymphocytes require qualitatively different signals from CD4(+) and CD8(+) T cells that most likely are provided to dendritic cells (DCs). This raises the question of whether the two T cell subsets are equally able to deliver the initial activation signal to DCs. Using ovalbumin as a model antigen we show that naive CD4(+) T cells cannot activate immature DCs and do not become activated, even though they recognize antigen on immature DCs. In contrast, naive CD8(+) T cells rapidly activate DCs and subsequently start to proliferate. This suggests that CD8(+) T cells contribute to DC activation prior to CD4(+) T cells and implies that CD8(+) T cells can provide help to CD4(+) T cells.     

Interleukin-1 inhibits renin gene expression in As4.1 cells but not in native juxtaglomerular cells

 Cardiovascular effects of inflammatory interleukins (IL) have been suggested to be mediated by the renin-angiotensin system in vivo. To address the direct cellular effect of IL, we examined the influence of IL-1β on renin secretion and renin mRNA in cultures of mouse juxtaglomerular granular (JG) cells and in the mouse tumor cell line As4.1, which expresses renin mRNA. Renin mRNA levels and secretion of active renin were not significantly changed by IL-1β in native JG cells. Activation of adenylyl cyclase by forskolin increased renin secretion and renin mRNA levels three- and fivefold, respectively. These stimulatory responses to forskolin were not altered by IL-1β. In contrast to native JG cells, renin mRNA abundance was markedly suppressed by IL-1β in As4.1 cells, whereas secretion of active renin and the stability of renin mRNA were not changed. In As4.1 cells forskolin did not change renin secretion or renin mRNA abundance in the absence or in the presence of IL-1β. These findings suggest that IL-1β has no direct influence on renin secretion and renin mRNA abundance at the level of native JG cells. Received: 15 December 1997 / Received after revision: 25 April 1998 / Accepted: 27 May 1998     

Ly49Q ligand expressed by activated B cells induces plasmacytoid DC maturation

Ly49Q, a type II C‐type lectin expressed on mouse plasmacytoid DC (pDC), contains a single carbohydrate recognition domain in its extracellular region and an ITIM in its cytoplasmic domain. We have identified the MHC class I molecule H‐2K^b as a Ly49Q ligand, confirming prior reports. Although H‐2K^b is expressed on essentially all hematopoietic cells, we found that only CpG‐stimulated B cells were able to activate Ly49Q. This discovery correlated with our finding that although H‐2K^b forms clusters on CpG‐activated B cells, it is diffusely expressed on resting B cells. Furthermore, CpG‐stimulated, but not resting, B cells up‐regulated co‐stimulatory molecules on pDC. This finding was confirmed by the fact that binding by anti‐Ly49Q mAb to Ly49Q led to pDC maturation in vitro. Our results suggest that clustered H‐2K^b on activated B cells act as ligands for Ly49Q and induce pDC maturation in vitro.     

Provocation with adenosine 5''-monophosphate, but not methacholine, induces sputum eosinophilia

INTRODUCTION: Bronchial hyper-responsiveness is usually measured with direct stimuli such as methacholine (MCh) or histamine. Adenosine 5'-monophosphate (AMP), which acts indirectly via the secondary release of mediators, is another stimulus to measure bronchial hyper-responsiveness. AIM: To investigate whether provocation with inhaled AMP itself initiates an inflammatory response resulting in an influx of eosinophils into the airway lumen. METHODS: We have included 21 non-smoking atopic asthmatic subjects (mean FEV1 101% predicted, mean age 34 years). Each subject performed three sputum inductions on different days, at least seven days apart: one without previous provocation, one hour after PC20 methacholine, and one hour after PC20 AMP. RESULTS: After provocation with AMP, but not methacholine, the percentage of sputum eosinophils increased significantly (from 1.9+/-0.5% to 4.5+/-1% (P<0.01) and 1.9+/-0.5% (P=0.89)). No changes in the percentages of neutrophils, lymphocytes, macrophages, or bronchial epithelial cells were found. CONCLUSION: A provocation test with AMP leads to an increased percentage of sputum eosinophils. This observation cannot be explained by a non-specific response of the airways to a vigorous bronchoconstriction, since methacholine had no effect on inflammatory cells.     

Interferon-gamma inhibits the proliferation but not the differentiation of murine B cells in response to IL-5

Interferon-gamma (IFN-gamma) is supposed to be produced by type 1 helper T cells (TH1) and inhibits IL-4-dependent B cell growth and differentiation. IL-5 (T cell-replacing factor, TRF), is a T cell-derived lymphokine which is predominantly produced by type 2 helper T cells (TH2) and regulates proliferation and differentiation of activated B cells. In this study, the effect of IFN-gamma on IL-5-dependent B cell growth and differentiation has been studied using murine chronic B cell leukemic cells (BCL1), normal splenic B cells, and cloned early B cell line. IFN-gamma selectively inhibits the IL-5-mediated proliferation of activated B cells as well as cloned early B cell lines at a low concentration (2 U/ml) in which polyclonal IgM production was not affected. This inhibitory effect of IFN-gamma occurs within 24 h after the onset of culture, as demonstrated by the inability of antibody to IFN-gamma to reverse totally the IFN-gamma-mediated suppressive effects if it was added later than 24 h after the onset of the culture. On the contrary, IL-5-mediated IgM secretion of BCL1 and IgA formation of LPS-stimulated normal B cells were relatively resistant to the suppressive effect of IFN-gamma. IFN-gamma does not affect the receptor expression for IL-5. Interestingly, IL-4-mediated IgG1 formation of LPS-stimulated B cells was markedly suppressed by IFN-gamma at 10 U/ml. These results strongly suggest that IFN-gamma may have differential effects on IL-5-mediated B cell triggering.     

Interleukin-5 is essential for vaccine-mediated immunity but not innate resistance to a filarial parasite

The study of protective immune mechanisms effective against filarial nematodes has been hampered by the inability of these important human pathogens to infect laboratory mice. Recently, Litomosoides sigmodontis, a natural parasite of rats, has been developed as a valuable model for the study of filarial infection. BALB/c mice are fully susceptible to infection with L. sigmodontis third-stage larvae and develop patent infection. In contrast, mice on the C57BL background are resistant, and parasites undergo only a single molt and do not mature to adulthood. We used interleukin-5 (IL-5)-deficient mice on the C57BL/6 background to address the role of IL-5 and eosinophils in the innate resistance of C57BL/6 mice. We found no differences in parasite survival between IL-5-deficient and C57BL/6 mice. However, when these mice were used for the analysis of vaccine-mediated immunity, a critical role for IL-5 was elucidated. Mice genetically deficient in IL-5 were unable to generate a protective immune response when vaccinated with irradiated larvae, whereas C57BL/6 mice were fully protected from challenge infection. These studies help to clarify the highly controversial role of eosinophils in filarial infection.     

In vivo notch reactivation in differentiating cochlear hair cells induces sox2 and prox1 expression but does not disrupt hair cell maturation

COVER PHOTOGRAPH: Lineage tracing of Prox1 expressing inner ear cochlear cells in Prox1CreEGFP/+; Rosa26‐EYFPloxp/+ mice at P23. All cochlear hair cells are labeled with calbindin in red. EYFP in green historically records the cochlear cells experiencing Prox1 expression during the development, which include hair cells and supporting cells (both inside and outside the organ of Corti). From Liu et al., Developmental Dynamics 241:684–696, 2012.     

Phosphorylcholine on isologous red blood cells induces polyclonal but not anti-phosphorylcholine plaque-forming cells in mice

It has been demonstrated in the preceding report (Bach, M. A., Beckmann, E. and Levitt, D., Eur. J. Immunol. 1984. 14: 589) that phosphorylcholine (PC) on the bacterium Streptococcus pneumoniae R36a stimulated polyclonal as well as anti-PC plaque-forming cells (PFC) in mouse spleen in vivo. In this study, red blood cells from BALB/c mice (MRBC) were either conjugated with PC, 2,4,6-trinitrophenyl (TNP) or treated with phospholipase A2 (PLA2) to expose PC on the cell membrane (determined by hemagglutination with the anti-PC myeloma HOPC8). When BALB/c mice were immunized i.v. with the conjugated or enzyme-treated MRBC, a significant polyclonal antibody response occurred (p less than 0.05) using PC-MRBC or PLA2-treated MRBC, but not with TNP-MRBC or sham-treated MRBC. No anti-PC or anti-MRBC immunoglobulin-secreting cells developed after immunization. Repeated immunization with PC-MRBC resulted in similar levels of protein A PFC after each immunization but no anti-PC, anti-MRBC or anti-PC-MRBC PFC. Thus, PC on R36a or isologous RBC stimulated increased numbers of splenic plaque-forming cells. In the case of R36a, 10-25% of these PFC produced antibodies directed towards PC. In contrast, PC-MRBC or PLA2-treated MRBC, failed to evoke any anti-PC antibody responses.     

5-氮胞苷诱导体外培养的胚胎干细胞向心肌样细胞分化

目的研究5-氮胞苷(5-aza)体外诱导胚胎干细胞分化为心肌细胞的可行性。方法将P19细胞接种于培养板中或铺有琼脂的培养板中,5μmol/L5-aza培养7d后用不含5-aza的培养基继续培养。倒置显微镜观察细胞跳动情况,用免疫细胞化学、RT-PCR检测及电镜观察等方法鉴定细胞分化。结果5-aza暴露结合悬浮培养可诱导P19细胞分化为有节律跳动的心肌细胞。分化的细胞表达心肌特异的GATA-4、α-MHC mRNA及α-sarcomeric actin、cTnT蛋白,同时透射电镜观察到细胞质内有明显的肌丝。结论5-aza在体外可诱导胚胎干细胞向心肌样细胞分化,悬浮培养有助于细胞的心肌化过程。