Alum-precipitated Ovalbumin Research Articles

CD8 T cells induce T-bet–dependent migration toward CXCR3 ligands by differentiated B cells produced during responses to alum-protein vaccines

Antibody-forming cells (AFCs) expressing the chemokine receptor CXCR3 are recruited to sites of inflammation where they help clear pathogens but may participate in autoimmune diseases. Here we identify a mechanism that induces CXCR3 expression by AFC and germinal center (GC) B cells. This happens when CD8 T cells are recruited into CD4 T cell-dependent B-cell responses. Ovalbumin-specific CD4 T cells (OTII) were transferred alone or with ovalbumin-specific CD8 T cells (OTI) and the response to subcutaneous alum-precipitated ovalbumin was followed in the draining lymph nodes. OTII cells alone induce T helper 2-associated class switching to IgG1, but few AFC or GC B cells express CXCR3. By contrast, OTI-derived IFN-γ induces most responding GC B cells and AFCs to express high levels of CXCR3, and diverse switching to IgG2a, IgG2b, with some IgG1. Up-regulation of CXCR3 by GC B cells and AFCs and their migration toward its ligand CXCL10 are shown to depend on B cells' intrinsic T-bet, a transcription factor downstream of the IFN-γR signaling. This model clarifies how precursors of long-lived AFCs and memory B cells acquire CXCR3 that causes their migration to inflammatory foci.

Helios Is Associated with CD4 T Cells Differentiating to T Helper 2 and Follicular Helper T Cells In Vivo Independently of Foxp3 Expression

BackgroundAlthough in vitro IL-4 directs CD4 T cells to produce T helper 2 (Th2)-cytokines, these cytokines can be induced in vivo in the absence of IL-4-signalling. Thus, mechanism(s), different from the in vitro pathway for Th2-induction, contribute to in vivo Th2-differentiation. The pathway for in vivo IL-4-independent Th2-differentiation has yet to be characterized.FindingsHelios (ikzf2), a member of the Ikaros transcription regulator family, is expressed in thymocytes and some antigen-matured T cells as well as in regulatory T cells. It has been proposed that Helios is a specific marker for thymus-derived regulatory T cells. Here, we show that mouse ovalbumin-specific CD4 (OTII) cells responding to alum-precipitated ovalbumin (alumOVA) upregulate Th2 features - GATA-3 and IL-4 - as well as Helios mRNA and protein. Helios is also upregulated in follicular helper T (TFh) cells in this response. By contrast, OTII cells responding to the Th1 antigen - live attenuated ovalbumin-expressing Salmonella - upregulate Th1 features - T-bet and IFN-γ - but not Helios. In addition, CD4 T cells induced to produce Th2 cytokines in vitro do not express Helios. The kinetics of Helios mRNA and protein induction mirrors that of GATA-3. The induction of IL-4, IL-13 and CXCR5 by alumOVA requires NF-κB1 and this is also needed for Helios upregulation. Importantly, Helios is induced in Th2 and TFh cells without parallel upregulation of Foxp3. These findings suggested a key role for Helios in Th2 and TFh development in response to alum-protein vaccines. We tested this possibility using Helios-deficient OTII cells and found this deficiency had no discernable impact on Th2 and TFh differentiation in response to alumOVA.ConclusionsHelios is selectively upregulated in CD4 T cells during Th2 and TFh responses to alum-protein vaccines in vivo, but the functional significance of this upregulation remains uncertain.

IFN-γ produced by CD8 T cells induces T-bet–dependent and –independent class switching in B cells in responses to alum-precipitated protein vaccine

Alum-precipitated protein (alum protein) vaccines elicit long-lasting neutralizing antibody responses that prevent bacterial exotoxins and viruses from entering cells. Typically, these vaccines induce CD4 T cells to become T helper 2 (Th2) cells that induce Ig class switching to IgG1. We now report that CD8 T cells also respond to alum proteins, proliferating extensively and producing IFN-γ, a key Th1 cytokine. These findings led us to question whether adoptive transfer of antigen-specific CD8 T cells alters the characteristic CD4 Th2 response to alum proteins and the switching pattern in responding B cells. To this end, WT mice given transgenic ovalbumin (OVA)-specific CD4 (OTII) or CD8 (OTI) T cells, or both, were immunized with alum-precipitated OVA. Cotransfer of antigen-specific CD8 T cells skewed switching patterns in responding B cells from IgG1 to IgG2a and IgG2b. Blocking with anti-IFN-γ antibody largely inhibited this altered B-cell switching pattern. The transcription factor T-bet is required in B cells for IFN-γ-dependent switching to IgG2a. By contrast, we show that this transcription factor is dispensable in B cells both for IFN-γ-induced switching to IgG2b and for inhibition of switching to IgG1. Thus, T-bet dependence identifies distinct transcriptional pathways in B cells that regulate IFN-γ-induced switching to different IgG isotypes.

Molecular differences between the divergent responses of ovalbumin-specific CD4 T cells to alum-precipitated ovalbumin compared to ovalbumin expressed by Salmonella

CD4 T helper (Th) cell differentiation defined by in vitro cytokine-directed culture systems leaves major gaps in our knowledge of the mechanisms driving divergent Th differentiation. This is evident from our analysis of the response of mouse ovalbumin-specific CD4 T cells to different forms of ovalbumin that induce markedly distinct responses in vivo. We show that live attenuated ovalbumin-expressing Salmonella (SalOVA) induce Th1-associated T-bet and IFN-γ. Conversely, alum-precipitated ovalbumin (alumOVA) induces the Th2-associated GATA-3 and IL-4. The early diversity occurring within these CD4 T cells isolated 3 days after immunization was assessed using real-time RT-PCR microfluidic cards designed with 384 selected genes. The technique was validated both at the population and single cell levels at different stages of the responses, showing β2-microglobulin to be a more stably expressed reference mRNA than either β-actin or 18S RNA. SalOVA was then shown selectively to induce the OVA-specific CD4 T cells to produce many chemokines and pro-inflammatory cytokines, contrasting with alumOVA-induced cells that only produced a few Th2-associated cytokines. Several cytokines and features associated with follicular helper functions were induced in the OVA-specific CD4 T cells by both antigens. Finally, IL-17RB is strongly associated with OVA-specific CD4 T cells responding to alumOVA, suggesting that alum may promote Th2 immune response through a role for the IL-25/IL-17RB pathway.

Modulation of Oral Tolerance to Ovalbumin by Dietary Protein in Mice

This study sought to determine whether oral tolerance to ovalbumin (OVA), responsible for food allergy, is affected by different amounts of protein intake. For this, 6-wk-old BALB/c mice were fed with low protein (5%, LP), normal protein (20%, NP) and high protein (40%, HP) diets, orally given either OVA (OVA-fed) or water (Water-fed) for 4 d, and then immunized intraperitoneally twice at a 3-wk interval with alum-precipitated OVA. After the last immunization, sera were collected to measure total and OVA-specific IgE by enzyme assay (ELISA). Splenocytes were cultured and stimulated with concanavalin A (Con A), lipopolysaccharide (LPS) or OVA and assayed for 3H-thymidine incorporation. The culture supernatants from their splenocytes stimulated with OVA were analyzed for interleukin (IL)-4, interferon (IFN)-gamma, and IL-12. Total IgE was significantly higher in OVA-fed HP groups as compared to NP and LP groups (p<0.05). The highest and the lowest OVA-specific IgE were observed in HP and LP diet groups, respectively (p<0.05). OVA-fed mice receiving the LP diet demonstrated significantly lower IL-4 as compared to the other two groups (p<0.05), while IFN-gamma was significantly higher in the LP compared to the HP group (p<0.05). Levels of IL-12 did not differ among the OVA-fed groups. Splenocytes of OVA-fed mice kept on the LP and HP diet showed significant impairment of proliferation to OVA as compared to the NP group (p<0.01). Proliferation against Con A was impaired in the LP group compared to the NP group (p<0.05) but not in Water-fed groups. However, it was higher against LPS in the HP than the LP group (p<0.05) both in Water-fed and OVA-fed animals. All these findings indicate that established oral tolerance to OVA is clearly affected by the amount of protein diet. They support the suggestion that dietary protein plays an important role(s) in IgE-mediated food allergies.

Suppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the high-affinity antibody response in C57BL/6 mice.

In the humoral immune response to an invasion of foreign antigens, B cells differentiate into low-affinity antibody-forming cells (AFCs) that mainly secrete IgM or, through germinal center (GC) formation, into high-affinity AFCs that secrete IgG-class antibodies with a higher affinity for the antigen. Previous studies have established the suppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on low-affinity antibody responses to antigens. However, whether and how TCDD affects the high-affinity antibody response to antigens has not yet been clarified. In this paper we investigate the effects of TCDD on GC formation, high-affinity AFC generation, and high-affinity antibody production in the primary humoral immune response. C57BL/6 mice were orally administered 0 or 20 microg/kg of TCDD and subsequently immunized with alum-precipitated ovalbumin (OVA) on day 0. Then the GC formation in the spleen and OVA-specific antibodies in the plasma, was evaluated until day 14 postimmunization. TCDD exposure reduced the production of OVA-specific IgG1 on days 10 and 14. GC formation in the spleen was also suppressed by TCDD exposure, and the suppression persisted from day 7 until day 14. In TCDD-administered mice, on day 7, cellular proliferation in the GCs was significantly suppressed, although apoptosis was not markedly affected. In order to measure high-affinity antibody and high-affinity AFCs, the mice were administered TCDD followed by immunization with alum-precipitated (4-hydroxy-3-nitrophenyl) acetyl linked to chicken gamma-globulin (NP-CG). The frequency of high-affinity NP-specific AFCs that bind to low-haptenated antigen was clearly shown to be reduced in the spleen on days 10 and 14. Furthermore, the high-affinity anti-NP IgG1 levels on days 10 and 14 postimmunization were significantly reduced by TCDD exposure. Taken together, the results of this paper demonstrate that TCDD exposure inhibits the generation of high-affinity AFCs and high-affinity antibody production during the primary humoral immune response and suggest that these alterations were caused by the suppression of antigen-responding B-cell proliferation induced by TCDD during GC formation.

Antigen‐specific CD8+ T cells inhibit IgE responses and interleukin‐4 production by CD4+ T cells

There is a growing body of evidence which suggests that CD8+ T cells play an important part in regulating the IgE response to non-replicating antigens. In this study we have systematically investigated their role in the regulation of IgE and of CD4+ T cell responses to ovalbumin (OVA) by CD8+ T cell depletion in vivo. Following intraperitoneal immunization with alum-precipitated OVA, OVA-specific T cell responses were detected in the spleen and depletion of CD8+ T cells in vitro significantly enhanced the proliferative response to OVA. Depletion of CD8+ T cells in vivo 7 days after immunization failed to enhance IgE production, while depletion of CD8+ T cells on days 12-18 greatly enhanced the IgE response, which rose to 26 micrograms/ml following a second injection of anti-CD8 on day 35 and remained in excess of 1 microgram/ml over 300 days afterwards. Reconstitution on day 21 of rats CD8-depleted on day 12 with purified CD8+ T cells from animals immunized on day 12 completely inhibited the IgE response. This effect was antigen specific; CD8+ T cells from OVA-primed animals had little effect on the IgE response of bovine serum albumin immunized rats. In vivo, CD8+ T cell depletion decreased interferon (IFN)-gamma production but enhanced interleukin (IL)-4 production by OVA-stimulated splenic CD4+ T cells. Furthermore, CD8+ T cell depletion and addition of anti-IFN-gamma antibody enhanced IgE production in vitro in an IL-4-supplemented mixed lymphocyte reaction. These data clearly show that antigen-specific CD8+ T cells inhibit IgE in the immune response to non-replicating antigens. The data indicate two possible mechanisms: first, CD8+ T cells have direct inhibitory effects on switching to IgE in B cells and second, they inhibit OVA-specific IL-4 production but enhance IFN-gamma production by CD4+ T cells.

IgE and mast cell responses on intestinal allergen exposure: A murine model to study the onset of food allergy

Objective: Allergic reactions to food are characterized by enhanced allergen-specific IgE serum levels and the activation of intestinal mast cells. Here we describe a murine model for the onset of food allergy and the role of cytokines in the regulation of food-induced IgE responses. Methods: Mice were primed systemically with low doses of alum-precipitated ovalbumin. Subsequent intragastric challenge led to enhanced sensitization. Results: Compared with baseline ovalbumin-specific IgE levels before challenge (0.23 ± 0.06 optical density [OD] units), ovalbumin-challenged mice showed significantly elevated IgE levels (0.86 ± 0.23 OD units) after intragastric challenge, which were not observed in control animals (0.29 ± 0.06 OD units). IgE levels mirrored intestinal mast cell activation, measured by decreased histamine levels in duodenal specimens, in ovalbumin-challenged mice (92.6 ± 7.9 ng/0.1 gm tissue weight) but not in saline-challenged mice (135.4 ± 18.3 ng/0.1 gm tissue weight), compared with baseline levels (141.1 ± 4.1 ng/0.1 gm tissue weight). Changes in IgE and histamine levels after intragastric challenge could be blocked by treating the animals with neutralizing antibodies against IL-4 or IL-10. Although it is generally accepted that ingestion of food allergens leads to a state of immunologic unresponsiveness (i.e., oral tolerance), it is shown here that low-dose systemic priming followed by intragastric challenge leads to sensitization instead of unresponsiveness. Conclusions: Our murine model shows an important correlation between T h2 cytokines, IgE production, and histamine release. Hence, this in vivo model provides a useful tool with which the complex mechanism underlying sensitization to food allergens can be studied. (J Allergy Clin Immunol 1997;99:94-9.)

Involvement of IL-5 in a murine model of allergic pulmonary inflammation: prophylactic and therapeutic effect of an anti-IL-5 antibody.

Interleukin-5 (IL-5) is important in the control of differentiation, migration, and activation of eosinophils. In order to study the role of IL-5 in the development of eosinophilic inflammation of the airways, we have used a monoclonal antibody to murine IL-5 (TRFK-5) in a murine model of allergic pulmonary inflammation. B6D2F1 mice were sensitized with alum-precipitated ovalbumin and were challenged with aerosolized ovalbumin on day 12 after sensitization. Samples of bronchoalveolar lavage (BAL) fluid, lung tissue, blood, and bone marrow aspirate were collected at different times after ovalbumin challenge. Twenty-four hours after challenge there were significant increases in the number of eosinophils in the BAL fluid, lung tissue, and blood while bone marrow eosinophils were decreased. Treatment of sensitized mice with TRFK-5 (0.01-1 mg/kg, i.p.) 2 h before ovalbumin challenge reduced the numbers of eosinophils in the BAL fluid and lung tissue and prevented the decrease in bone marrow eosinophils in a dose-dependent fashion. The number of eosinophils in the BAL fluid, peribronchial and alveolar regions of the lung was also reduced when TRFK-5 (2 mg/kg, i.p.) was given up to 5 d after ovalbumin challenge. Furthermore, there was no evidence of increased epithelial damage, edema, or the presence of mucus that could have resulted from eosinophil apoptosis and release of toxic proteins after neutralization of IL-5. These results demonstrate an important role for IL-5 in the development of eosinophilic inflammation of the airways and for the migration of eosinophils from the bone marrow into blood in response to antigen challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Mast cells modulate allergic pulmonary eosinophilia in mice.

Mast cells are important effector cells in IgE-mediated acute allergic reactions. Mast cells also produce cytokines such as interleukin (IL)-3, IL-4, IL-5, tumor necrosis factor (TNF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) that regulate the function of eosinophils and the development of a late-phase inflammatory response to antigen challenge. To evaluate the role of mast cells on the development of IgE-mediated allergic pulmonary eosinophilia in vivo, we compared the eosinophil infiltration into lungs of mast cell deficient mice (WBB6F1/J-W/Wv) with their congenic normal littermates (W/W+). Mice were sensitized with alum-precipitated ovalbumin and challenged with aerosolized ovalbumin on day 12 after sensitization. Bronchoalveolar lavage (BAL) fluid, lung tissue biopsies, and blood samples were collected after ovalbumin challenge. Eosinophil numbers in the BAL and lung tissue, lung eosinophil peroxidase (EPO) activity and serum levels of IgE and IgG1 were measured. In sensitized W/W+ mice, there were increased numbers of eosinophils in the BAL fluid and lung tissue, and EPO levels were increased after ovalbumin challenge. Ovalbumin challenge of sensitized mast-cell-deficient mice produced fewer numbers of eosinophils in the BAL fluid and lungs, and EPO levels were also reduced compared with their challenged congenic littermates. On the other hand, levels of serum IgE and IgG1 were not different between W/Wv mice and their congenic littermates.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a murine model of allergic pulmonary inflammation.

Pulmonary inflammation with eosinophil (EOs) infiltration is a prominent feature of allergic respiratory diseases such as asthma. In order to study the cellular response during the disease development, an animal model of IgE-mediated pulmonary inflammation with characteristic eosinophilia is needed. We developed a method for inducing severe pulmonary eosinophilia in the mouse and also studied the numbers of EOs in blood and bone marrow and the response to corticosteroid treatment. Animals were sensitized with alum-precipitated ovalbumin (OVA) and challenged with aerosolized OVA 12 days later when serum IgE levels were significantly elevated. Four to eight hours after challenge there were moderate increases in the number of EOs in the bone marrow and peripheral blood, but only a few EOs were observed in the lung tissue and in bronchoalveolar lavage (BAL) fluid. Twenty-four hours after challenge, there was a marked reduction of EOs in bone marrow, while the number of EOs peaked in the perivascular and peribronchial regions of the lung. Forty-eight hours after challenge, the highest number of EOs was found in the BAL fluid, making up > 80% of all cells in that compartment. The high levels of EOs in the lung tissue and BAL fluid lasted for 2-3 days and was followed by a more moderate but persistent eosinophilia for another 10 days. Nonsensitized animals showed no significant changes in the number of EOs in BAL fluid, lungs, blood or bone marrow. Histopathological evaluation also revealed epithelial damage, excessive mucus in the lumen and edema in the submucosa of the airways.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of pertussis toxin on delayed-type hypersensitivity responses and on the activity of suppressor T cells on the responses

Experiments were performed on mice to investigate the effects of pertussis toxin (PT) on delayed-type hypersensitivity (DTH) to ovalbumin (OA) and on the activity of suppressor T cells on the DTH (DTH-Ts). Mice immunized with alum-precipitated ovalbumin showed a transient DTH, which was determined as footpad swelling which disappeared 2 weeks after immunization. Maximal footpad swelling was observed 24 hr after DTH elicitation. On the other hand, when mice received PT (2 μg/mouse) at the time of immunization, the transient DTH became an enhanced and persistent DTH, which persisted for at least 4 weeks. In addition, the time of maximum footpad swelling was delayed from 24 to 48 hr after DTH elicitation. The immune spleen T cells from PT-treated mice showed a persistently high ability to transfer DTH into syngenic naive mice. DTH-Ts was induced in spleens of mice injected iv with OA-coupled syngeneic spleen cells. However, when these mice received PT at the time of suppressor induction, their spleen cells revealed considerably reduced suppressor activity. The activity of DTH-Ts was also reduced when DTH-Ts were either treated in vitro with PT or transferred into PT-injected recipient mice. From these results, interference with the suppressor function of DTH-Ts from PT was considered to be, at least in part, as an enhancing mechanism of DTH.

Adoptive transfer of "persistent' IgE responses in mice in the absence of secondary antigenic stimulation.

Spleen cells were prepared from Balb/c mice immunized 30 days previously with alum-precipitated ovalbumin (OA), which manifested high, persistent titres of anti-OA IgE and IgG. The adoptive transfer of 5.0 X 10(7) such cells to X-irradiated syngeneic recipients produced comparable persistent IgE/IgG responses, in the absence of secondary antigenic challenge. Fractionation procedures indicated that the nylon-wool adherent population from the spleen was the most active in effecting transfer of the response. However, donor T-cells were also required, as pretreatment of the cellular inoculum with anti-Thy 1.2 antiserum ablated transfer. The inclusion of serum containing anti-OA IgG (but not IgE) in the cellular inoculum also blocked the transfer.

Preferential synthesis of IgE reaginic antibodies in rats immunized with alum-adsorbed antigens.

The reaginic antibody response to alum-precipitated ovalbumin (OA) and the dialyzed water-soluble extracts of ragweed (DWSR) and Alternaria tenuis (DWST) in several strains of rats appeared to be wholly an IgE response. There was no evidence of a heat-stable (IgGa) antibody to OA, DWSR and DWST in the sera of the rats immunized with these antigens suspended in alum. Wistar-Furth and Lew inbred and hooded outbred rats produced comparable amounts of reaginic antibody after immunization with DWST, but BN inbred rats failed to generate a reaginic response to this antigen. The amount of antigen-induced histamine release from rat peritoneal mast cells did not always correlate with the level of circulating IgE-specific antibody.

Complement in the Induction of IgA and IgE Antibody Production

Abstract The IgA-antibody forming cell response to sheep erythrocytes in BALB/c and CBA mice depleted of complement by cobra factor (CoF) was suppressed to 10 to 40% of the level in controls. The IgE response to alum-precipitated ovalbumin was measured in NIH mice by passive cutaneous anaphylaxis in rat skin. Reaginic antibody was scarcely detectable in CoF-treated animals whereas controls had titers of up to 1:2000. Fourteen days after boosting with ovalbumin, control mice had peripheral eosinophilia whereas animals which had received CoF before priming 6 weeks earlier had levels only minimally raised above normal. Studies of the generation of T cell blasts, “education” of thymus cells, and priming of splenic T cells indicated that activation of T cells by antigen proceeded normally under conditions in which thymus-dependent antibody production was suppressed by in vivo complement depletion. Pure sheep IgG anti-mouse C3 and pure F(ab′)2 anti-mouse C3 antibody both inhibited the in vitro T-dependent antihapten response of mouse spleen cells to TNP-KLH.

Heterogeneity of Rabbit Anti-Ovalbumin Antibodies Sensitizing Human, Guinea Pig and Rabbit Skin

Summary Three groups of rabbits were injected with five-times recrystallized ovalbumin. One group received soluble ovalbumin, the second alumprecipitated ovalbumin, and the third ovalbumin in complete Freund's adjuvant. The developmental courses of human, guinea pig and rabbit skin-sensitizing antibodies were followed, and representative sera were fractionated by electrophoresis and by Sephadex G 200. The effect of heating at 56°C for 4 hr was also studied. Rabbit anti-ovalbumin antisera are capable of sensitizing human, guinea pig and rabbit skin. A different antibody sensitizes the skin of each of the three species. Human SSA was best stimulated by antigen in Freund's adjuvant, was first detectable at 3 to 4 weeks and peaked at 10 to 12 weeks. It is only alightly affected by heat, is fast-moving electrophoretically and elutes with the ascending 7 S peak from Sephadex G 200. Guinea pig PCA antibody was produced effectively by each immunization technique used, appeared early and continued at high titer for several months. It is heat stable, is slow-moving electrophoretically and coincides with the 7 S peak from Sephadex G 200. Rabbit homologous PCA antibody was best stimulated by alum-precipitated ovalbumin, was demonstrable at 2 weeks and was usually sustained for several months. This antibody is affected but not destroyed by heat, is fast-moving electrophoretically, and elutes with the ascending 7 S peak from Sephadex G 200. The GPPCA appears to coincide with the IgG immunoglobulin class, confirming the results of others (12, 13). The immunoglobulin class or classes of HSSA and RaPCA antibodies have not been identified in these studies, but are fast-moving electrophoretically and are perhaps slightly larger in size than IgG immunoglobulins. In this system, the P-K reaction is not comparable to PCA in the guinea pig.

Quantitative Studies in Anaphylaxis

Summary Alum-precipitated ovalbumin had a sensitizing capacity in guinea-pigs of from 4 to nearly 400 times greater than that of ovalbumin in saline solution, depending upon the route of administration of the sensitizing dose. For ovalbumin in saline solution the sensitizing capacity of the antigen by intraäbdominal administration was about 1½ times greater than by intravenous injection and nearly 2½ times greater that by subcutaneous administration. Only the latter difference was statistically significant. For alum-precipitated ovalbumin the sensitizing capacity by subcutaneous administration was 2½ times greater than by intraäbdominal administration and 60 times greater than by intravenous injection. Both of these differences were statistically significant.