Role of B cells in TH cell responses in a mouse model of asthma

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Effect of Fusobacterium nucleatum on the T and B cell responses to Porphyromonas gingivalis in a mouse model

T cell cytokine profiles and specific serum antibody levels in five groups of BALB/c mice immunized with saline alone, viable Fusobacterium nucleatum ATCC 25586, viable Porphyromonas gingivalis ATCC 33277, F. nucleatum followed by P. gingivalis and P. gingivalis followed by F. nucleatum were determined. Splenic CD4 and CD8 cells were examined for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma and IL-10 by dual colour flow cytometry and the levels of serum anti-F. nucleatum and anti-P. gingivalis antibodies determined by an ELISA. Both Th1 and Th2 responses were demonstrated by all groups, and while there were slightly lower percentages of cytokine positive T cells in mice injected with F. nucleatum alone compared with the other groups immunized with bacteria, F. nucleatum had no effect on the T cell production of cytokines induced by P. gingivalis in the two groups immunized with both organisms. However, the percentages of cytokine positive CD8 cells were generally significantly higher than those of the CD4 cells. Mice immunized with F. nucleatum alone had high levels of serum anti-F. nucleatum antibodies with very low levels of P. gingivalis antibodies, whereas mice injected with P. gingivalis alone produced anti-P. gingivalis antibodies predominantly. Although the levels of anti-F. nucleatum antibodies in mice injected with F. nucleatum followed by P. gingivalis were the same as in mice immunized with F. nucleatum alone, antibody levels to P. gingivalis were very low. In contrast, mice injected with P. gingivalis followed by F. nucleatum produced equal levels of both anti-P. gingivalis and anti-F. nucleatum antibodies, although at lower levels than the other three groups immunized with bacteria, respectively. Anti-Actinobacillus actinomycetemcomitans, Bacteroides forsythus and Prevotella intermedia serum antibody levels were also determined and found to be negligible. In conclusion, F. nucleatum immunization does not affect the splenic T cell cytokine response to P. gingivalis. However, F. nucleatum immunization prior to that of P. gingivalis almost completely inhibited the production of anti-P. gingivalis antibodies while P. gingivalis injection before F. nucleatum demonstrated a partial inhibitory effect by P. gingivalis on antibody production to F. nucleatum. The significance of these results with respect to human periodontal disease is difficult to determine. However, they may explain in part differing responses to P. gingivalis in different individuals who may or may not have had prior exposure to F. nucleatum. Finally, the results suggested that P. gingivalis and F. nucleatum do not induce the production of cross-reactive antibodies to other oral microorganisms.     

Kinetics of cellular and cytokine responses in a chimeric mouse model for the study of staphylococcal enterotoxin B pathogenesis

The mechanisms by which superantigens, such as staphylococcal enterotoxin B (SEB), contribute to microbial pathogenicity have been poorly defined. The study of such pathogenic processes has been hampered by the lack of an adequate animal model. We utilized a previously described murine chimeric model to determine the cytokines and cell populations that might be involved in SEB toxicity. In the absence of bone marrow transplantation (BMT), all total body irradiated (TBI) mice died, while all transplanted mice survived up to 6 months. Compared with non-TBI and non-BMT mice, chimeric mice had an increased percentage of CD11b (Mac-1)-positive splenocytes (17 vs. 59%, P < 0.05) and decreased CD45R-positive (B) cells (33 vs. 6%, P < 0.05) at 6 weeks after BMT. The relative numbers of splenocyte CD4 and CD8 cells were similar in chimeric and normal mice. Susceptibility of chimeric animals to 10 or 100 microg SEB was time-dependent: no mice challenged at 2 weeks post-BMT died, but 15% of mice challenged at 4 weeks and 50% of those challenged at 6-8 weeks died. Compared with TBI and non-BMT C3H/HeJ mice, SEB-challenged chimeric mice at 6-8 weeks had (1) increased splenocyte mRNA expression for: IFN-gamma (3.5 x optimally at 1 h), TNF-alpha (6.5 x at 2 h), IL-6 (4.8 x at 4 h), IL-1beta (8.4 x at 4 h), IL-2 (4.7 x at 4 h), and IL-10 (3 x at 16 h), and (2) increased and earlier peak serum levels of IFN-gamma, IL-6, IL-1beta and IL-2, but no increase in serum TNF-alpha or IL-4. These data support the hypothesis that the decreased percentage of B cells and increased macrophages in chimeric mice lead to enhanced T cell-macrophage interactions after SEB administration and a lethal burst of T cell and macrophage cytokine release. This model will provide insight into cell populations and mechanisms that mediate superantigen-induced toxicity.     

Human antibody responses to bacterial antigens: studies of a model conventional antigen and a proposed model B cell superantigen.

We have investigated the human antibody repertoires that bind to two different classes of bacterial antigens. Immunization with the conventional antigen, type b capsular polysaccharide of Haemophilus influenzae Hib PS, uniformly induces IgA and IgG responses dominated by clones that use heavy chains structurally related to two subsets of VH3 genes, while in a minority of subjects antibodies from the VH1 or VH4 families are co-induced. In contrast, the "alternative binding site" of Staphylococcal Protein A (SPA) represents an unconventional determinant, because; (i) SPA is bound by a large proportion of non-immune IgM, IgA and IgG F(ab')2, (ii) SPA is bound only to Fab from the VH3 family, which can be encoded by at least four different germline genes, (iii) SPA binding is independent of VL usage, (iv) by flow cytometry SPA is bound by > 15% of tonsilar B cells, but not to T cells. (v) In vitro stimulation with an SPA containing mitogen induces the preferentially production of Ig bearing a VH3 marker. Taken together, these studies characterize a VH family restricted binding interaction that is distinct from the properties associated with conventional antigens such as Hib PS. Based on these data we propose that SPA represents a prototype for a B cell superantigen.     

Inflammatory responses to amyloidosis in a transgenic mouse model of Alzheimer's disease

Mutations in the amyloid precursor protein (APP) and presenilin-1 and -2 genes (PS-1, -2) cause Alzheimer's disease (AD). Mice carrying both mutant genes (PS/APP) develop AD-like deposits composed of beta-amyloid (Abeta) at an early age. In this study, we have examined how Abeta deposition is associated with immune responses. Both fibrillar and nonfibrillar Abeta (diffuse) deposits were visible in the frontal cortex by 3 months, and the amyloid load increased dramatically with age. The number of fibrillar Abeta deposits increased up to the oldest age studied (2.5 years old), whereas there were less marked changes in the number of diffuse deposits in mice over 1 year old. Activated microglia and astrocytes increased synchronously with amyloid burden and were, in general, closely associated with deposits. Cyclooxygenase-2, an inflammatory response molecule involved in the prostaglandin pathway, was up-regulated in astrocytes associated with some fibrillar deposits. Complement component 1q, an immune response component, strongly colocalized with fibrillar Abeta, but was also up-regulated in some plaque-associated microglia. These results show: i) an increasing proportion of amyloid is composed of fibrillar Abeta in the aging PS/APP mouse brain; ii) microglia and astrocytes are activated by both fibrillar and diffuse Abeta; and iii) cyclooxygenase-2 and complement component 1q levels increase in response to the formation of fibrillar Abeta in PS/APP mice.     

TNFRSF13B in B cell responses to organ transplantation

Antibodies directed against organ transplants are thought to pose the most vexing hurdle to enduring function and survival of the transplants, particularly organ xenotransplants, and accordingly basic and clinical investigation has focused on elucidating the specificity and pathogenicity of graft-specific antibodies. While much has been learned about these matters, far less is known about the B cells producing graft-specific antibodies and why these antibodies appear to injure some grafts but not others. With the goal of addressing those questions, we have investigated the properties of tumor necrosis factor receptor super family-13B (TNFRSF13B), which regulates various aspects of B cell responses. A full understanding of the functions of TNFRSF13B however is hindered by extreme polymorphism and by diversity of interactions of the protein. Nevertheless, TNFRSF13B variants have been found to exert distinct impact on natural and elicited antibody responses and host defense and mutations of TNFRSF13B have been found to influence the propensity for development of antibody-mediated rejection of organ transplants. Because B cell responses potentially limit application of xenotransplantation, understanding how TNFRSF13B diversity and TNFRSF13B variants govern immunity in xenotransplantation could inspire development of novel therapeutics that could in turn accelerate clinical implementation of xenotransplantation.     

A multiple transgenic mouse model with a partially humanized activation pathway for helper T cell responses

Mice expressing human CD4 and human MHC II molecules provide a valuable model both for the investigation of the immunopathogenetic role of human autoantigens and for the development of therapeutic strategies based on modulating helper T cell activation in vivo. Here we present a novel mouse model expressing HLA-DR17 (a split antigen of HLA-DR3) together with human CD4 in the absence of murine cd4 (CD4/DR3 mice). Human CD4 accurately replaces murine cd4 within T cells. In particular, the preservation of cd8(+) and CD4(+) T cell subsets distinguishes CD4/DR3 mice from other multiple transgenic models in which the alternative T cell subsets are fundamentally disturbed. Moreover, human CD4 is also faithfully expressed on antigen presenting cells such as dendritic cells and monocyte/macrophages, so that the overall transgenic CD4 expression pattern resembles very closely that of humans. HLA-DR3 expression in the thymus correlates very closely to that of mouse MHC II. In contrast, only 70% of mouse MHC II positive cells in spleen, lymph node, and peripheral blood coexpress HLA-DR3. No significant bias was found with regard to particular leucocytes in this respect. The stimulation of helper T cells clearly depends on the interaction between the human transgene products, since mAbs to HLA-DR and/or CD4 completely blocked in vitro recall responses to tetanus toxoid. CD4/DR3 mice represent a partially humanized animal model which will facilitate studies of DR3-associated autoimmune responses and the in vivo determination of the therapeutic potential of mAbs to human CD4.     

Activation of a nonclassical NKT cell subset in a transgenic mouse model of hepatitis B virus infection

NKT cells are specialized cells of the immune system that bear both T cell and NK cell markers. Classical NKT cells display TCRs of restricted heterogeneity (Valpha14-Jalpha281) and recognize lipid antigens (e.g., alpha-galactosyl ceramide) presented by nonpolymorphic CD1 molecules. Recently, other nonclassical NKT subsets have been recognized, including NKT cells not reactive with CD1d-alpha-galactosyl ceramide complexes. The biological functions of these cells are unknown. Here, we show that nonclassical NKT cells that are CD1d restricted but nonreactive to alpha-GalCer are activated in response to hepatocytes expressing hepatitis B viral antigens in a transgenic mouse model of acute hepatitis B virus infection. Our results document the first in vivo function for such nonclassical NKT cells and suggest a role for these cells in the host response to HBV infection.     

Microglial responses to dopamine in a cell culture model of Parkinson's disease

Activated microglia appear to selectively attack dopamine (DA) neurons in the Parkinson's disease (PD) substantia nigra. We investigated potential mechanisms using culture models. As targets, human SH-SY5Y cells were left undifferentiated (UNDIFF) or were differentiated with retinoic acid (RA) or RA plus brain-derived neurotrophic factor (RA/BDNF). RA/BDNF-treated cells were immunoreactive for tyrosine hydroxylase and the DA transporter, took up exogenous DA, and released DA after K(+) stimulation. Undifferentiated and RA-treated cells lacked these characteristics of a DA phenotype. Co-culture of target cells with human elderly microglia resulted in elevated toxicity in DA phenotype (RA/BDNF) cells. Lipopolysaccharide (LPS) plus K(+)-stimulated DA release enhanced toxicity by 500-fold. DA induced microglial chemotaxis in Boyden chambers. Spiperone inhibited this effect. Cultured human elderly microglia expressed mRNAs for D1-D4 but not D5 DA receptors. The microglia, as well as PD microglia in situ, were also immunoreactive for D1-D4 but not D5 DA receptors. These findings demonstrate that activated microglia express DA receptors, and suggest that this mechanism may play a role in the selective vulnerability of DA neurons in PD.     

播散性鼠B细胞淋巴瘤动物模型的建立

目的 在具有免疫能力的BALB/c小鼠上构建播散性B淋巴瘤动物模型.方法 经尾静脉注射鼠源性B细胞淋巴瘤细胞株A20于同源BALB/c小鼠,0～3个月间观察动物成瘤情况;取动物脏器行石蜡包埋、病理切片、HE染色;流式细胞仪检测其CD19的表达.结果 尾静脉注射2×106、2×107细胞于14只BALB/c小鼠,成瘤时间分别为76.8±12.0天和26.1±7.9天;总体成瘤率分别为 71.4%(5/7)和100%(7/7);在肝脏、脾脏、淋巴结、肠、肠系膜、下肢、颈部、子宫和臀部等多脏器成瘤;流式检测瘤组织细胞mCD19表达强阳性.结论运用尾静脉注射方法构建了内脏器官广泛发生的播散性B淋巴瘤BALB/c小鼠动物模型,为进一步进行B淋巴瘤相关研究提供了实验依据.     

Marginal zone B cell enrichment and strong follicular B cell reduction correlate with a delayed IgG response in a light chain diversity restricted mouse model

Recently developed B6.kappa(-)lambda(SEG) mice (by crossing kappa(-) and C57BL/6 mice congenic for the wild Mus spretus SEG strain lambda locus lacking genes coding for lambda1 and lambda3) have a very reduced light chain diversity. B6.kappa(-)lambda(SEG) mice produce only lambda2 and lambdax light chains. Regardless of their Igh haplotype, B6.kappa(-)lambda(SEG) mice show a restricted B cell distribution by light chain subtype with lambdax dominance in all peripheral compartments except peritoneal cavity where lambda2 is dominant. This distribution suggests that selection mechanisms act differently in different B cell compartments on lambda2 and lambdax bearing B cells. Sequence analysis before or following immunization did not reveal unusual mechanisms of diversification. B6.kappa(-)lambda(SEG) mice still respond to various challenging antigens using new Ab patterns. In particular, regardless of Igh(a) or Igh(b) haplotypes, the anti-2,4-dinitrophenyl response is characterized by a restricted diversity for both heavy and light chains and a delayed IgG response when compared to B6 and B6.kappa(-) mice. We suggest that the delayed IgG response is due to the expansion of marginal zone B cells whereas follicular B cells are strongly reduced.     

Allelic variation linked to adenine phosphoribosyltransferase locus in mouse teratocarcinoma cell line and feral-derived mouse strains

Southern blot analysis reveals two distinct adenine phosphoribosyltransferase (APRT)alleles in the P-19 mouse teratocarcinoma cell line. One allele is identical to that observed in common laboratory mouse strains (Mus musculus domesticus).The restriction enzyme site variations between the two alleles occur in sequences located both upstream and downstream of the APRTgene, but not within it. Although the P-19 cell line was established from a C3H strain embryo (Mus musculus domesticus),a sixth generation ancestor of this embryo was a feral mouse (Mus musculus musculus).The restriction pattern of the variant APRTallele in P-19 is identical to that of a feral-derived Mus musculus musculusanimal, establishing the origin of this allele in the P-19 cell line. A third, distinct APRTallele was found in a Mus spretusferal-derived mouse. Exploiting the differences between the two APRTalleles in the P-19 cell line, we have demonstrated their sequential loss in APRT-deficient clones.     

B‐cell anergy induces a Th17 shift in a novel B lymphocyte transgenic NOD mouse model,the 116C‐NOD mouse

Autoreactive B lymphocytes play a key role as APCs in diaebetogenesis. However, it remains unclear whether B‐cell tolerance is compromised in NOD mice. Here, we describe a new B lymphocyte transgenic NOD mouse model, the 116C‐NOD mouse, where the transgenes derive from an islet‐infiltrating B lymphocyte of a (8.3‐NODxNOR) F1 mouse. The 116C‐NOD mouse produces clonal B lymphocytes with pancreatic islet beta cell specificity. The incidence of T1D in 116C‐NOD mice is decreased in both genders when compared with NOD mice. Moreover, several immune selection mechanisms (including clonal deletion and anergy) acting on the development, phenotype, and function of autoreactive B lymphocytes during T1D development have been identified in the 116C‐NOD mouse. Surprisingly, a more accurate analysis revealed that, despite their anergic phenotype, 116C B cells express some costimulatory molecules after activation, and induce a T‐cell shift toward a Th17 phenotype. Furthermore, this shift on T lymphocytes seems to occur not only when both T and B cells contact, but also when helper T (Th) lineage is established. The 116C‐NOD mouse model could be useful to elucidate the mechanisms involved in the generation of Th‐cell lineages.     

T and B cell responses to HDM allergens and antigens

House dust mites provide well-characterized proteins to study human responses to inhaled antigens. Even in the absence of allergy they induce a high frequency of T cell precursors. The healthy response manifests by T cell proliferation and Th1 cytokines with little antibody. Responses of allergic people include Th1 and Th2 cytokines and IgE, IgG1, and IgG4 antibodies. Regulatory cells limit effector responses in healthy people. About half the IgE and IgG antibodies bind the group 1 and 2 allergens and 30% bind the group 4, 5, and 7 allergens. Although HLA independent, the recognition of the group 1 allergen shows an immunodominant region and a T cell receptor bias. The major allergens are not produced in higher amounts than many of the poorly non-allergenic proteins. The non-allergenic mite ferritin antigen shows high T cell proliferative responses with mixed cytokine production.     

NK cell depletion diminish tumour-specific B cell responses

Natural killer (NK) cells can exercise immediate cytotoxicity against malignant cells and thus far modulate the development of tumour directed T cell immunity. To investigate the impact of NK cells on the development of tumour directed B cell immunity mice were immunised with IMR5-75 human neuroblastoma cells with or without prior in vivo NK cell depletion. Flow cytometry analyses gave evidence for an impaired IgG response against the cells immunised with. Dissection of Th1 (IgG2a) and Th2 (IgG1) oriented B cell responses revealed Th1 responses as primarily affected, while Th2 oriented B cell responses as measured by flow cytometry and GD2 ganglioside-specific ELISA were enforced. The data reveal an unexpected impact of NK cells on the development of tumour directed B cell responses. Consequently, NK cell function has also to be taken into account when developing B cell-based cancer immunotherapy.     

Innate control of B cell responses

Mature B cells generate protective immunity by undergoing immunoglobulin (Ig) class switching and somatic hypermutation, two Ig gene-diversifying processes that usually require cognate interactions with T cells that express CD40 ligand. This T cell-dependent pathway provides immunological memory but is relatively slow to occur. Thus, it must be integrated with a faster, T cell-independent pathway for B cell activation through CD40 ligand-like molecules that are released by innate immune cells in response to microbial products. Here, we discuss recent advances in our understanding of the interplay between the innate immune system and B cells, particularly at the mucosal interface. We also review the role of innate signals in the regulation of Ig diversification and production.