ABSTRACT
H-/K-Ras and N-Ras isoforms were proposed to lack functional specificities due to similarity in 1-165 amino acids. As recent studies implied Ras isoform-specific developmental effects, we examined their functional specificity using Leishmania major infection, anti-hapten antibody response and carrier-specific T cell response. While N-Ras overexpression increased L. major infection in resistant C57BL/6 mice, H-Ras or K-Ras overexpression reduced the infection in susceptible BALB/c mice. These Ras isoforms differentially regulated anti-TNP antibody response in TNP-Ova-primed, but not in TNP-Ficoll- or TNP-LPS-primed, BALB/c mice. Ras isoform-specific silencing selectively modulated Ova-specific T cell response. The data indicate Ras isoform-specific regulation of antigen-specific immune response.
Subject(s)
Leishmaniasis, Cutaneous/immunology , Protein Isoforms/immunology , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/immunology , Animals , Antibody Formation/immunology , Antigen-Antibody Reactions/immunology , Cell Line , Female , Ficoll/analogs & derivatives , Ficoll/immunology , Haptens/immunology , Leishmania major/immunology , Leishmaniasis, Cutaneous/pathology , Lymphocyte Activation/immunology , Macrophages/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Ovalbumin/immunology , Protein Isoforms/genetics , RNA Interference , RNA, Small Interfering/genetics , T-Lymphocytes, Regulatory/immunology , Trinitrobenzenes/immunologyABSTRACT
We examined the effect of nobiletin (5,6,7,8,3',4'-hexamethoxyflavone) on immune response in ovalbumin (OVA)-immunized mice. Treatment with nobiletin increased OVA-specific IL-4 and IL-10 production. In addition, mice that received nobiletin showed higher levels of OVA-specific IgE, IgG and IgG1 production than did control mice. The antibody response to the thymus-independent antigen 2,4,6-trinitrophenyl-Ficoll was not different in the control and nobiletin groups, suggesting that nobiletin does not directly stimulate antibody production. An in vitro study showed that treatment with nobiletin enhanced the ability of antigen presentation of bone marrow-derived dendritic cells. The in vivo and in vitro results indicate that nobiletin regulates immune function.
Subject(s)
Flavones/pharmacology , Immunity, Cellular , Immunity, Humoral , Ovalbumin/immunology , Animals , Bone Marrow Cells/drug effects , Bone Marrow Cells/immunology , Cells, Cultured , Cytokines/immunology , Cytokines/metabolism , Dendritic Cells/drug effects , Dendritic Cells/immunology , Ficoll/analogs & derivatives , Ficoll/immunology , Immunity, Cellular/drug effects , Immunity, Cellular/immunology , Immunity, Humoral/drug effects , Immunity, Humoral/immunology , Immunization , Mice , Mice, Inbred BALB C , Trinitrobenzenes/immunologyABSTRACT
Cytotoxic T cells (CTLs) are important immune effector cells in the adaptive immune response. It has been well documented that CTLs are important in host immune responses to viral and bacterial intracellular pathogens, tumors, and transplanted tissues. The properties of CTLs have been studied extensively in murine models, and their roles validated in the human setting. Frequently, the presence of these cells correlates well with protective immunity, so the ability to readily measure the activity of these cells is an important immunological measurement. In this unit, several assays are described that are commonly utilized to induce CTLs and to measure CTL activity both in vitro and in vivo. These assays are adaptable to many experimental and/or disease models, and in the case of the in vitro assays can be applied to measure CTL activity in human samples. © 2018 by John Wiley & Sons, Inc.
Subject(s)
T-Lymphocytes, Cytotoxic/immunology , Animals , Antigens, Viral/immunology , Chromium , DNA Fragmentation , Mice , Minor Histocompatibility Antigens/immunology , Trinitrobenzenes/immunology , Virus Diseases/immunologyABSTRACT
Immune-mediated drug hypersensitivity reactions (IDHR) may result from immuno-sensitization to a drug-induced neo-antigen. They rarely occur in patients and are usually not predicted preclinically using standard toxicity studies. To assess the potential of a drug to induce T-cell sensitization, trinitrophenyl (TNP)-Ficoll was used here as a bystander antigen in animal experiments. TNP-Ficoll will only elicit TNP-specific IgG antibodies in the presence of non-cognate T-cell help. Therefore, the presence of TNP-specific IgG antibodies after co-injection of drug and TNP-Ficoll was indicative of T-cell sensitization potential. This TNP-Ficoll-approach was used here to characterize T-cell help induced by oral exposure to diclofenac (DF) or carbamazepine (CMZ). DF or CMZ was administered orally to BALB/c mice and after 3 w, the mice were challenged in a hind paw with TNP-Ficoll and a dose of the drug that by itself does only elicit a sub-optimal popliteal lymph node assay (PLNA) response. T-cell-dependent responses were then evaluated in paw-draining popliteal lymph nodes (PLN). Also, shortly after oral exposure, mesenteric lymph nodes (MLN) were excised for evaluation of local responses. Both drugs were able to increase PLN cellularity and TNP-specific IgG1 production after challenge. Both DF and CMZ stimulated CD4+ and CD8+ T-cells and caused shifts of the subsets toward an effector phenotype. DF, but not CMZ, appeared to stimulate interferon (IFN)-γ production. Remarkably, depletion of CD8+, but not CD4+, T-cells reduced TNP-specific IgG1 production, and was more pronounced in CMZ- than in DF-exposed animals. Local responses in the MLN caused by DF or CMZ also showed shifts of CD4+ and CD8+-cells toward a memory phenotype. Together, the data indicate that oral exposure to CMZ and DF differentially induced neo-antigen-specific T-cell reactions in the PLNA.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Carbamazepine/adverse effects , Diclofenac/adverse effects , Drug Hypersensitivity/immunology , T-Lymphocytes/immunology , Administration, Oral , Animals , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Antigens/immunology , Carbamazepine/therapeutic use , Diclofenac/therapeutic use , Ficoll/analogs & derivatives , Ficoll/immunology , Immunoglobulin G/metabolism , Interferon-gamma/metabolism , Lymph Nodes/pathology , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Trinitrobenzenes/immunologyABSTRACT
LRRK2 is the causal molecule of autosomal dominant familial Parkinson's disease. B2 cells express a much higher LRRK2 mRNA level than B1 cells. To reveal the function of LRRK2 in B cells, we analyzed B cell functions in LRRK2-knockout (LRRK2(-/-)) mice. LRRK2(-/-) mice had significantly higher counts of peritoneal B1 cells than wild-type mice. After BCR stimulation, phosphor-Erk1/2 of splenic B2 cells was enhanced to a higher degree in LRRK2(-/-) mice. LRRK2(-/-) mice had a significantly higher serum IgA level, and TNP-Ficoll immunization increased the titer of serum anti-TNP IgM antibody. LRRK2 may play important roles in B cells.
Subject(s)
B-Lymphocytes/metabolism , Homeostasis/genetics , Immunoglobulin A/blood , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/physiology , Age Factors , Animals , Antigens, CD/metabolism , B-Lymphocytes/classification , Enzyme-Linked Immunosorbent Assay , Ficoll/analogs & derivatives , Ficoll/immunology , Flow Cytometry , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , MAP Kinase Signaling System/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Peritoneal Cavity/cytology , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/metabolism , Signal Transduction/genetics , Spleen/cytology , Transforming Growth Factor beta1/blood , Trinitrobenzenes/immunologyABSTRACT
Tumour necrosis factor receptor-associated factor 3 (TRAF3) interacting protein 3 (TRAF3IP3; also known as T3JAM) is expressed specifically in immune organs and tissues. To investigate the impact of TRAF3IP3 on immunity, we generated Traf3ip3 knock-out (KO) mice. Interestingly, these mice exhibited a significant reduction in the number of common lymphoid progenitors (CLPs) and inhibition of B cell development in the bone marrow. Furthermore, Traf3ip3 KO mice lacked marginal zone (MZ) B cells in the spleen. Traf3ip3 KO mice also exhibited a reduced amount of serum natural antibodies and impaired T cell-independent type II (TI-II) responses to trinitrophenol (TNP)-Ficoll antigen. Additionally, our results showed that Traf3ip3 promotes autophagy via an ATG16L1-binding motif, and MZ B cells isolated from mutant mice showed a diminished level of autophagy and a high rate of apoptosis. These results suggest that TRAF3IP3 contributes to MZ B cell survival by up-regulating autophagy, thereby promoting the TI-II immune response.
Subject(s)
Autophagy/genetics , B-Lymphocytes/immunology , Carrier Proteins/immunology , Gene Expression Regulation , Membrane Proteins/immunology , Animals , Apoptosis/genetics , Apoptosis/immunology , Autophagy/immunology , Autophagy-Related Proteins , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Differentiation/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Ficoll/immunology , HeLa Cells , Humans , Lymphocyte Activation/immunology , Lymphoid Progenitor Cells/immunology , Lymphoid Tissue/immunology , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Spleen/cytology , Spleen/immunology , T-Lymphocytes/immunology , Trinitrobenzenes/immunology , Up-RegulationABSTRACT
Murine contact sensitivity (CS) reaction could be antigen-specifically regulated by T CD8(+) suppressor (Ts) lymphocytes releasing microRNA-150 in antibody light-chain-coated exosomes that were formerly suggested to suppress CS through action on macrophages (Mφ). The present studies investigated the role of Mφ in Ts cell-exosome-mediated antigen-specific suppression as well as modulation of Mφ antigen-presenting function in humoral and cellular immunity by suppressive exosomes. Mice depleted of Mφ by clodronate liposomes could not be tolerized and did not produce suppressive exosomes. Moreover, isolated T effector lymphocytes transferring CS were suppressed by exosomes only in the presence of Mφ, demonstrating the substantial role of Mφ in the generation and action of Ts cell regulatory exosomes. Further, significant decrease of number of splenic B cells producing trinitrophenyl (TNP) -specific antibodies with the alteration of the ratio of serum titres of IgM to IgG was observed in recipients of exosome-treated, antigen-pulsed Mφ and the significant suppression of CS was demonstrated in recipients of exosome-treated, TNP-conjugated Mφ. Additionally, exosome-pulsed, TNP-conjugated Mφ mediated suppression of CS in mice pre-treated with a low-dose of cyclophosphamide, suggesting de novo induction of T regulatory (Treg) lymphocytes. Treg cell involvement in the effector phase of the studied suppression mechanism was proved by unsuccessful tolerization of DEREG mice depleted of Treg lymphocytes. Furthermore, the inhibition of proliferation of CS effector cells cultured with exosome-treated Mφ in a transmembrane manner was observed. Our results demonstrated the essential role of Mφ in antigen-specific immune suppression mediated by Ts cell-derived exosomes and realized by induction of Treg lymphocytes and inhibition of T effector cell proliferation.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Exosomes/immunology , Immune Tolerance/immunology , Macrophages/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Cell Proliferation , Cells, Cultured , Cyclophosphamide/pharmacology , Dermatitis, Contact/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Immunosuppression Therapy/methods , Immunosuppressive Agents/pharmacology , Lymphocyte Depletion , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , MicroRNAs/genetics , Trinitrobenzenes/immunologyABSTRACT
Crosslinking of the immunoglobulin receptor FcεRI activates basophils and mast cells to induce immediate and chronic allergic inflammation. However, it remains unclear how the chronic allergic inflammation is regulated. Here, we showed that ecto-nucleotide pyrophosphatase-phosphodiesterase 3 (E-NPP3), also known as CD203c, rapidly induced by FcεRI crosslinking, negatively regulated chronic allergic inflammation. Basophil and mast cell numbers increased in Enpp3(-/-) mice with augmented serum ATP concentrations. Enpp3(-/-) mice were highly sensitive to chronic allergic pathologies, which was reduced by ATP blockade. FcεRI crosslinking induced ATP secretion from basophils and mast cells, and ATP activated both cells. ATP clearance was impaired in Enpp3(-/-) cells. Enpp3(-/-)P2rx7(-/-) mice showed decreased responses to FcεRI crosslinking. Thus, ATP released by FcεRI crosslinking stimulates basophils and mast cells for further activation causing allergic inflammation. E-NPP3 decreases ATP concentration and suppresses basophil and mast cell activity.
Subject(s)
Adenosine Triphosphate/metabolism , Asthma/immunology , Basophils/immunology , Mast Cells/immunology , Phosphoric Diester Hydrolases/immunology , Pyrophosphatases/immunology , Receptors, IgE/immunology , Adenosine Triphosphate/pharmacology , Animals , Basophils/cytology , Dermatitis, Contact/immunology , Diarrhea/immunology , Diarrhea/pathology , Immunoglobulin E/immunology , Mast Cells/cytology , Mice , Mice, Inbred BALB C , Mice, Knockout , Passive Cutaneous Anaphylaxis/immunology , Phosphoric Diester Hydrolases/genetics , Pyrophosphatases/genetics , RNA Interference , RNA, Small Interfering , Receptors, Purinergic P2X7/genetics , Receptors, Purinergic P2X7/immunology , Trinitrobenzenes/immunologyABSTRACT
Immunoglobulins protect against disease to a considerable extent by activating complement and stimulatory immunoglobulin crystallizable fragment receptors (Ig FcRs), and aggregating microbial pathogens. Yet IgG1, the predominant murine serum Ig isotype, cannot activate complement by the classical pathway, binds more avidly to an inhibitory than to stimulatory FcRs, and has limited ability to aggregate pathogens. In these regards, it resembles human IgG4 (ref. 4). We hypothesized that limited ability to activate effector mechanisms might protect against immune complex immunopathology. Here we show that IgG1-deficient (γ1(-)) mice, immunized with a potent antigen, develop lethal renal disease soon after they begin to produce antigen-specific antibody, whereas similarly immunized wild-type mice remain healthy. Surprisingly, renal disease in this model is complement and FcR independent and results from immune complex precipitation in glomerular capillaries, as in some cryoglobulinaemic humans. IgG3, which self-associates to form large immune complexes, accounts for more than 97% of the mouse Ig in this cryoglobulin; furthermore, glomerular disease develops when mice are injected with IgG3 anti-trinitrophenyl (TNP) monoclonal antibody followed by a TNP-labelled protein. Renal disease is prevented in both active and passive immunization models by antigen-specific IgG1; other isotypes are less potent at preventing disease. These observations demonstrate the adaptive significance of Ig isotypes that poorly activate effector mechanisms, reveal an immune-complex-dependent, complement- and FcR-independent nephrotoxic mechanism, and suggest that isotypes that poorly activate effector mechanisms may be useful for inhibiting immune complex immunopathology.
Subject(s)
Cryoglobulinemia/complications , Glomerulonephritis/etiology , Glomerulonephritis/prevention & control , Immunoglobulin G/immunology , Animals , Antibodies, Monoclonal/immunology , Antigen-Antibody Complex/chemistry , Antigen-Antibody Complex/immunology , Antigens/immunology , Binding, Competitive , Complement System Proteins , Cryoglobulinemia/immunology , Cryoglobulinemia/pathology , Disease Models, Animal , Female , Glomerulonephritis/immunology , Glomerulonephritis/pathology , Goats , Male , Mice , Receptors, IgG , Solubility , Trinitrobenzenes/immunologyABSTRACT
BACKGROUND: Our previous work showed that epicutaneous (EC) immunization with protein antigen e.g. TNP-conjugated mouse immunoglobulin (TNP-Ig) in the form of a patch prior to hapten sensitization inhibits Th1-mediated contact hypersensitivity (CHS) in mice. We also found that suppression of CHS was mediated by TCRαß+ CD4+ CD8+ T suppressor cells producing TGF-ß. The aim of this study was to investigate the role of innate immunity in the suppression of CHS. METHODS: Mice were immunized by applying gauze patches containing protein antigen alone or in the presence of zymosan, and were then tested for the CHS response. Adoptive cell transfer experiments were used to study the mechanisms involved in the reversal of skin-induced suppression. The influence of EC immunization on cytokine production by lymph node cells was measured by ELISA. RESULTS: We found that EC immunization with TNP-Ig and zymosan before trinitrophenyl chloride sensitization reverses skin-induced suppression, demonstrated in vivo and in vitro. The reversal of skin-induced suppression was transferable by antigen-specific TCRαß+ CD4+ T contrasuppressor cells. Furthermore, we showed that the contrasuppression was IL-17A-dependent and TLR2- and MyD88-independent. CONCLUSIONS: Our work strongly suggests that EC immunization with protein antigen and zymosan reverses skin-induced suppression and that this approach may be a potential tool to increase the immunogenicity of weakly immunogenic antigens.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Immunoglobulins/immunology , Interleukin-17/immunology , Receptors, Antigen, T-Cell, alpha-beta/immunology , Skin/immunology , Trinitrobenzenes/immunology , Zymosan/immunology , Administration, Cutaneous , Animals , Antigens/immunology , Dermatitis, Contact/immunology , Haptens/immunology , Immunity, Innate/immunology , Immunization/methods , Immunosuppression Therapy/methods , Lymph Nodes/immunology , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Myeloid Differentiation Factor 88/immunology , Toll-Like Receptor 2/immunology , Transforming Growth Factor beta/immunology , Vaccination/methodsABSTRACT
Ag administered together with specific IgG3 induces a higher Ab response than Ag administered alone, an effect requiring the presence of complement receptors 1 and 2 (CR1/2). In this study, we have investigated the fate of Ag, the development of germinal centers (GCs), and the Ab response after i.v. administration of IgG3 anti-trinitrophenyl (TNP) in complex with OVA-TNP. After 2 h, OVA-TNP was detected on marginal zone (MZ) B cells, and a substantial amount of Ag was detected in splenic follicles and colocalized with follicular dendritic cells (FDCs). After 10 d, the percentage of GCs and the IgG responses were markedly higher than in mice immunized with uncomplexed OVA-TNP. The effects of IgG3 were dependent on CR1/2 known to be expressed on B cells and FDCs. Using bone marrow chimeric mice, we demonstrate that an optimal response to IgG3-Ag complexes requires that CR1/2 is expressed on both cell types. These data suggest that CR1/2(+) MZ B cells transport IgG3-Ag-C complexes from the MZ to the follicles, where they are captured by FDCs and induce GCs and IgG production. This pathway for initiating the transport of Ags into splenic follicles complements previously known B-cell dependent pathways where Ag is transported by 1) MZ B cells, binding large Ags-IgM-C complexes via CR1/2; 2) recirculating B cells, binding Ag via BCR; or 3) recirculating B cells, binding IgE-Ag complexes via the low-affinity receptor for IgE, CD23.
Subject(s)
Antigen-Antibody Complex/immunology , B-Lymphocytes/immunology , Dendritic Cells, Follicular/immunology , Immunoglobulin G/immunology , Spleen/immunology , Animals , Antigens/immunology , Female , Fingolimod Hydrochloride , Germinal Center/immunology , Immunoglobulin E/immunology , Immunoglobulin M/immunology , Immunosuppressive Agents/pharmacology , Lymphocyte Activation/immunology , Mice , Mice, Inbred BALB C , Mice, Knockout , Ovalbumin/immunology , Propylene Glycols/pharmacology , Receptors, Complement 3d/biosynthesis , Receptors, Complement 3d/genetics , Receptors, Complement 3d/immunology , Sphingosine/analogs & derivatives , Sphingosine/pharmacology , Spleen/cytology , Trinitrobenzenes/immunologyABSTRACT
BACKGROUND: Epicutaneous (EC) immunization offers a new method of a needle-free and self-administrable immunization by using a topically applied patch to deliver vaccine. We have previously shown that EC immunization with hapten-conjugated protein antigen TNP-Ig prior to hapten sensitization inhibits Th1-mediated contact hypersensitivity (CHS) in mice. Our further work showed that EC immunization with TNP-Ig and Toll-like receptor (TLR) ligands prior to hapten sensitization reverses skin-induced suppression. METHODS: Animal model of contact hypersensitivity was used to study reversal of skin-induced suppression. RESULTS: Current work showed that EC immunization with protein antigen TNP-Ig and MDP NOD2 agonist - muramyldipeptide (L isoform) reverses skin-induced suppression of CHS. On the other hand L18-MDP NOD2 agonist - muramyldipeptide with a C18 fatty acid chain and MDP control - negative control for MDP - muramyldipeptide (D isoform, inactive) did not reverse skin-induced suppression. "Transfer in" experiment showed that reversal of skin-induced suppression can be adoptively transferred with lymphoid cells isolated from donors EC treated with TNP-Ig and MDP NOD2 agonist. Moreover, experiment employing two non-cross-reacting antigens TNP-Ig and OX-Ig proved that reversal of skin-induced suppression is antigen specific. Additionally, lymph node cells isolated from mice EC immunized with TNP-Ig and MDP NOD2 agonist produced increased level of IFN-γ suggesting that this cytokine might be involved in reversal of skin-induced suppression. CONCLUSION: This work shows that EC immunization with protein antigen plus NOD2 ligand MDP may be a potential tool to increase the immunogenicity of weekly immunogenic antigens.
Subject(s)
Acetylmuramyl-Alanyl-Isoglutamine/immunology , Dermatitis, Contact/prevention & control , Immunization , Immunoglobulins/immunology , Nod2 Signaling Adaptor Protein/agonists , Skin/immunology , Trinitrobenzenes/immunology , Animals , Cytokines/biosynthesis , Male , Mice , Mice, Inbred CBAABSTRACT
Ab responses to T cell-independent type 2 (TI-2) Ags, such as bacterial capsular polysaccharides, are critical for host defense. In mice, B-1b cells expressing a CD11b(+)FSC(hi)CD21(lo/-)CD19(hi) phenotype play a key role in producing Abs against TI-2 Ags. In primates, a distinct IgM(+)CD27(+) "memory" B cell population is thought to generate TI-2 Ab responses, and evidence for a B-1b-like cell population participating in these responses is lacking. In this article, we demonstrate that nonhuman primates (NHPs; African green monkeys and cynomolgus macaques) harbor serosal B cells expressing a CD11b(+)FSC(hi)CD21(lo/-)CD80(+/-)CD19(hi) phenotype, constitutively active Stat3, and increased reactivity with phosphorylcholine, similar to murine peritoneal B-1a and B-1b cell populations. Like what is observed for murine B-1b cells, NHP CD11b(+)FSC(hi)CD21(lo/-)CD19(hi) B cells dominate the Ag-specific B cell response and Ab production against the TI-2 Ag trinitrophenyl-Ficoll. Although Ag-specific IgM(+) B cells expressing CD27 were not detected prior to immunization, Ag-specific CD11b(+)CD19(hi) B cells expressed and maintained an IgM(+)IgD(lo)CD27(+)CD80(+) phenotype following immunization. Thus, the murine and NHP B cell populations responding to trinitrophenyl-Ficoll are highly similar, with the main exception being that Ag-specific NHP B-1-like cells express CD27 following TI-2 Ag encounter. Therefore, murine B-1b and primate IgM(+)CD27(+) "memory" B cell subsets proposed to produce TI-2 Ab responses may be highly related, if not identical. Overall, these data not only support that B-1-like cells are present in NHPs but also provide evidence that these cells perform the same functions attributed to murine B-1b cells.
Subject(s)
Antigens/immunology , B-Lymphocyte Subsets/immunology , Epitopes/immunology , T-Lymphocytes/immunology , Animals , Antibody Formation/immunology , B-Lymphocyte Subsets/metabolism , Cells, Cultured , Chlorocebus aethiops , Ficoll/immunology , Macaca fascicularis , Male , Mice , Spleen/immunology , Spleen/metabolism , T-Lymphocytes/metabolism , Trinitrobenzenes/immunology , Tumor Necrosis Factor Receptor Superfamily, Member 7/metabolismABSTRACT
Dibenzo[def,p]chrysene (DBC) is a potent environmental carcinogen in rodents, fish, and human cells examined in culture. There are numerous similarities between the patterns of cytochrome P-450 (P450) activation of DBC and its covalent binding to DNA and proteins with another polycyclic aromatic hydrocarbon (PAH), 7,12-dimethylbenz[a]anthracene (DMBA). Our lab has previously shown that DMBA produces immunosuppression in rodents and human cell systems. Therefore, the purpose of these studies was to examine the immunotoxicity of DBC in a rodent model that was found to be sensitive to the immunosuppressive effects of DMBA. Data showed that DBC had similar potency to DMBA in producing suppression of a T-dependent antibody response (TDAR) and altered spleen cell subsets in a similar manner as DMBA when DMBA was given by gavage for 5 d in corn oil to mice at doses of 1-100 mg/kg total cumulative doses. T-cell-independent antigen (TNP-Ficoll) responses were quantitatively less sensitive to DBC suppression. It was also found that as with DMBA, DBC produced a persistent immunosuppression, which lasted for at least 4 wk following dosing with a novel pill method for self-administration of DBC. In conclusion, DBC appears to possess many of the same characteristics of DMBA in terms of its immunotoxicity.
Subject(s)
Antibody Formation/drug effects , Benzopyrenes/toxicity , Carcinogens, Environmental/toxicity , Spleen/drug effects , Spleen/immunology , Administration, Oral , Animals , Antibody Formation/immunology , Benzopyrenes/administration & dosage , Biomarkers , Carcinogens, Environmental/administration & dosage , Dose-Response Relationship, Drug , Ficoll/analogs & derivatives , Ficoll/immunology , Male , Membrane Proteins , Mice , Mice, Inbred C57BL , Spleen/cytology , Trinitrobenzenes/immunologyABSTRACT
Understanding the mechanisms that maintain protective antibody levels after immunisation is important for vaccine design. In this study, we have determined the kinetics of plasma and memory B cells detectable in the blood of cattle immunised with model T-dependent or T-independent antigens. Immunisation with the T-D antigen resulted in an expansion of TNP-specific plasma cells post-TNP primary and booster immunisations, which was associated with increased titres of TNP-specific IgG antibodies. Although no TNP-specific memory B cells were detected in the T-D group following the primary immunisation, we detected an increase in the number of TNP-specific memory B cells post-TNP boost. In contrast, no TNP-specific plasma or memory B cells were detected after primary or secondary immunisation with the T-I antigen. We then investigated if immunisation with a third party antigen (tetanus toxin fragment C, TTC) would result in a bystander stimulation and increase the number of TNP-specific plasma and memory B cells in the T-D and/or T-I group. TTC immunisation in the T-D group resulted in a small increase in the number of TNP-specific plasma cells post-TTC primary immunisation and boost, and in an increase in the number of TNP-specific memory B cells post-TTC boost. This bystander effect was not observed in the animals previously immunised with the T-I antigen. In conclusion, the present study characterised for the first time the B cell response in cattle to immunisation with T-D and T-I antigens and showed that bystander stimulation of an established T-D B cell memory response may occur in cattle.
Subject(s)
B-Lymphocytes/immunology , Cattle/immunology , Immunologic Memory , Plasma Cells/immunology , T-Lymphocytes/immunology , Animals , Antibodies/blood , Antigens/blood , Cattle/blood , Enzyme-Linked Immunospot Assay/veterinary , Ficoll/analogs & derivatives , Ficoll/immunology , Haptens/immunology , Immunoassay/veterinary , Leukocytes, Mononuclear/physiology , Male , Trinitrobenzenes/immunology , gamma-Globulins/immunologyABSTRACT
Phage display has enabled the rapid isolation of antigen-specific antibodies from combinatorial libraries of V(H) and V(L) genes obtained from lymphocytes of immunized animals. Recently, a different approach to antibody isolation that circumvents library screening and instead relies on the mining of the V(H) and V(L) gene repertoires obtained by high throughput sequencing of cDNAs from bone marrow antibody-secreting cells was reported. Here we compared the antibodies obtained via phage library screening or via repertoire mining of V gene cDNAs obtained from total splenocytes of mice immunized with the hapten trinitrophenyl (TNP) conjugated to carrier proteins. We show that, despite the large heterogeneity of B lymphocytes in the spleen, the most abundant V genes encoded antigen-specific antibodies, indicating that total splenocytes can be used in place of bone marrow plasma cells for antibody discovery at least in high titer animals. While both phage display and repertoire mining yielded antigen-specific antibodies showing comparable affinities by enzyme-linked immunosorbent assay analysis, clones obtained by the latter approach displayed higher selectivity towards TNP relative to control haptens. Interestingly, the antibody genes isolated by phage display were of low abundance or absent from the V gene repertoire obtained by 454 sequencing. Similarly, the highly abundant V genes identified by repertoire mining, that as soluble antibodies were antigen-specific, were found to be poorly displayed on phage and were not enriched by phage panning. Thus, our results reveal that phage display and repertoire mining of immune repertoires are complementary technologies that can yield different antigen-specific antibody clones.
Subject(s)
Antibodies/genetics , Gene Library , Haptens/immunology , Immunoglobulin Variable Region/genetics , Peptide Library , Trinitrobenzenes/immunology , Amino Acid Sequence , Animals , Antibodies/chemistry , Antibodies/immunology , Antibody Affinity , DNA, Complementary/genetics , Female , Immunization , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/immunology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Sequence Alignment , Spleen/cytologyABSTRACT
BACKGROUND: The clinical manifestations of food allergy include diarrhea and systemic anaphylaxis (shock), which can occur together or by themselves in different subjects. Although ingested food antigens need to be absorbed to induce shock, it is not known whether they need to be absorbed to induce diarrhea. OBJECTIVE: We sought to identify mechanisms that determine whether food allergy induces diarrhea versus shock and determine whether diarrhea requires absorption of ingested antigens. METHODS: These issues were studied in mice in active, passive, and hybrid immunization models. The active model was used to determine the allergic diarrhea susceptibility of J chain- and polymeric immunoglobulin receptor-deficient mice, which are unable to secrete IgA. The hybrid model was used to determine whether intravenously administered antigen-specific IgG antibody, which is not secreted into the gut, can protect against allergic diarrhea, as well as shock. RESULTS: Shock, but not diarrhea, was induced in naive mice by using intravenous IgE anti-trinitrophenyl (TNP) antibody, followed by oral TNP-BSA, whereas both were induced in mice presensitized with intraperitoneal ovalbumin/alum plus oral ovalbumin. More TNP-BSA was required to induce shock than diarrhea in presensitized mice, and intravenous IgG anti-TNP antibody, which is not secreted into the gut, protected these mice against both diarrhea and shock. Consistent with this, chicken ovalbumin-immunized J chain- and polymeric immunoglobulin receptor-deficient mice, which have high serum IgA levels but little intestinal IgA, resisted diarrhea induction. CONCLUSION: Intestinal immunity and oral antigen dose determine whether diarrhea, systemic anaphylaxis, or both are induced, and ingested antigen must be absorbed to induce either response.
Subject(s)
Anaphylaxis/immunology , Diarrhea/immunology , Food Hypersensitivity/immunology , Food Hypersensitivity/therapy , Immunoglobulins/therapeutic use , Anaphylaxis/etiology , Anaphylaxis/prevention & control , Animals , Clinical Protocols , Diarrhea/etiology , Diarrhea/prevention & control , Disease Models, Animal , Fatty Acid-Binding Proteins/genetics , Food Hypersensitivity/complications , Humans , Immunization , Immunoglobulin J-Chains/genetics , Immunoglobulins/administration & dosage , Immunoglobulins/metabolism , Interleukin-9/genetics , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, Transgenic , Ovalbumin/administration & dosage , Promoter Regions, Genetic/genetics , Receptors, IgG/genetics , Receptors, Polymeric Immunoglobulin/genetics , Serum Albumin, Bovine/administration & dosage , Serum Albumin, Bovine/immunology , Trinitrobenzenes/immunologyABSTRACT
14-3-3σ regulates cytokinesis and cell cycle arrest induced by DNA damage but its role in the immune system is unknown. Using gene-targeted 14-3-3σ-deficient (i.e., KO) mice, we studied the role of 14-3-3σ in B-cell functions. Total numbers of B cells were reduced by spontaneous apoptosis of peripheral B cells. Upon B-cell antigen receptor engagement in vitro, KO B cells did not proliferate properly or up-regulate CD86. In response to T cell-independent antigens, KO B cells showed poor secretion of antigen-specific IgM. This deficit led to increased lethality of KO mice after vesicular stomatitis virus infection. KO B cells showed elevated total FOXO transcriptional activity but also increased FOXO1 degradation. Coimmunoprecipitation revealed that endogenous 14-3-3σ protein formed a complex with FOXO1 protein. Our results suggest that 14-3-3σ maintains FOXO1 at a consistent level critical for normal B-cell antigen receptor signaling and B-cell survival.
Subject(s)
14-3-3 Proteins/immunology , B-Lymphocytes/immunology , Forkhead Transcription Factors/immunology , Homeostasis/immunology , 14-3-3 Proteins/genetics , 14-3-3 Proteins/metabolism , Adoptive Transfer , Animals , Antigens/immunology , Apoptosis/immunology , B-Lymphocytes/cytology , B-Lymphocytes/metabolism , Blotting, Western , Cell Proliferation , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Female , Ficoll/analogs & derivatives , Ficoll/immunology , Forkhead Box Protein O1 , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Immunoglobulin G/blood , Immunoglobulin M/blood , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Protein Binding , Receptors, Antigen, B-Cell/immunology , Reverse Transcriptase Polymerase Chain Reaction , Trinitrobenzenes/immunologyABSTRACT
BACKGROUND: TNFRSF13B, which encodes transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), is mutated in 10% of patients with common variable immunodeficiency. One of the 2 most common TACI mutations in common variable immunodeficiency, C104R, abolishes ligand binding and is found predominantly in the heterozygous state. The murine TACI mutant C76R is the equivalent of the human TACI mutant C104R. OBJECTIVE: We sought to define the consequence of the C76R mutation on TACI function in mice that express both wild-type TACI and the murine C76R mutant. METHODS: Transgenic mice that express murine TACI C76R, the counterpart of human TACI C104R, on the TACI(+/-) B6/129 background (C76R/TACI(+/-) mice) were constructed. Serum immunoglobulins and antibody responses to the type II T-independent antigen trinitrophenylated (TNP)-Ficoll were determined by means of ELISA. B-cell proliferation in response to a proliferation-inducing ligand was determined based on tritiated thymidine incorporation into DNA. IgG1 secretion by B cells in response to a proliferation-inducing ligand plus IL-4 was determined by means of ELISA. RESULTS: C76R/TACI(+/-) mice had significantly impaired antibody responses to the type II T-independent antigen TNP-Ficoll compared with TACI(+/+) B6/129 control animals, and their B cells were impaired in their capacity to proliferate and secrete IgG1 in response to TACI ligation. Unexpectedly, TACI(+/-) mice had similarly impaired B-cell function as C76R/TACI(+/-) littermates. Impaired TACI function caused by haploinsufficiency was confirmed in TACI(+/-) mice on the C57BL/6 background. CONCLUSION: These results suggest that the human TACI mutant C104R might impair TACI function in heterozygotes through haploinsufficiency.
Subject(s)
B-Lymphocytes/metabolism , Common Variable Immunodeficiency/immunology , Haploinsufficiency/immunology , Mutant Proteins/metabolism , Transmembrane Activator and CAML Interactor Protein/metabolism , Animals , Antibody Formation/genetics , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Cell Proliferation , Cells, Cultured , Common Variable Immunodeficiency/genetics , Ficoll/analogs & derivatives , Ficoll/immunology , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutant Proteins/genetics , Mutant Proteins/immunology , Mutation/genetics , Transmembrane Activator and CAML Interactor Protein/genetics , Transmembrane Activator and CAML Interactor Protein/immunology , Trinitrobenzenes/immunologyABSTRACT
Inflammation is a crucial step in the development of allergic contact dermatitis. The primary contact with chemical allergens, called sensitization, and the secondary contact, called elicitation, result in an inflammatory response in the skin. The ability of contact allergens to induce allergic contact dermatitis correlates to a great extent with their inflammatory potential. Therefore, the analysis of the sensitizing potential of a putative contact allergen should include the examination of its ability and potency to cause an inflammation. In this study, we examined the inflammatory potential of different weak contact allergens and of the strong sensitizer 2,4,6-trinitrochlorobenzene (TNCB) in vitro and in vivo using the contact hypersensitivity model, the mouse model for allergic contact dermatitis. Cytokine induction was analysed by PCR and ELISA to determine mRNA and protein levels, respectively. Inflammation-dependent recruitment of skin-homing effector T cells was measured in correlation with the other methods. We show that the sensitizing potential of a contact allergen correlates with the strength of the inflammatory response. The different methods used gave similar results. Quantitative cytokine profiling may be used to determine the sensitizing potential of chemicals for hazard identification and risk assessment.