Characterization of immunoglobulin E responses in Balb/c mice against the major allergens of timothy grass (Phleum pratense) pollen

BACKGROUND: Grass pollen, such as that from timothy grass (Phleum pratense), represents a major cause of type I allergy. OBJECTIVE: To characterize the IgE immune response and to identify the major allergens eliciting an IgE response in a mouse model using pollen extract of P. pratense for sensitization, in order to assess analogies to human hyperreactivity and to gain information on the allergenic potential as determined by the IgE-reactivity kinetics of defined allergens. METHODS: Balb/c mice were sensitized with pollen extract or with purified natural allergens. Serum IgE levels, the induction of specific IgE antibodies and immediate hypersensitivity were monitored by ELISA, Western blot and a skin test, respectively. RESULTS: The sensitized mice mounted a strong IgE response and showed IgE-reactivity first against Phl p 5a and 5b, then Phl p 4 and 13 and lastly against Phl p 6. No IgE response was mounted against Phl p 1. However, all purified fractions examined (Phl p 5a, 5b, 6 and 1) induced specific IgE and showed similar kinetics of IgE induction as pollen extract (first Phl p 5a and 5b, then Phl p 6). Skin test experiments demonstrated positive reactivity only in sensitized mice. CONCLUSION: The IgE reactivity induced by the major allergens in Balb/c mice was very similar to that found in allergic patients, with the exception of Phl p 1. The kinetics of the specific IgE response was comparable using either pollen extract or the purified major allergens, indicating that the intrinsic properties of the allergens are of importance rather than their proportionate amounts in pollen extract. This model should prove to be suitable for investigations regarding the mechanisms of induction and manifestation of timothy grass pollen allergy and for the evaluation of therapeutic strategies.     

Complementary DNA cloning and expression of a newly recognized high molecular mass allergen Phl p 13 from timothy grass pollen (Phleum pratense)

BACKGROUND: Grass pollen extracts contain a range of different allergenic components that can be classified as having low, middle or high molecular mass. Almost 75% of patients allergic to grass pollen display immunoglobulin (Ig) E-reactivity to allergens in the high molecular mass range of 55-60 kDa. These proteins have not yet been fully characterized on the protein and DNA level. OBJECTIVE: The aim of this study was to identify and characterize an allergen of the high molecular mass fraction of Phleum pratense pollen by N-terminal protein sequencing and molecular cloning. METHODS: A previously uncharacterized allergen which migrates as a double band with a molecular mass of 55-60 kDa was biochemically purified and investigated by N-terminal sequencing. Subsequently, a DNA primer was designed to amplify the corresponding cDNA using PCR. The cloned cDNA and deduced amino acid sequence were compared with sequence data bases. Immunoblots carrying the recombinant expression product were developed with monoclonal antibodies and sera derived from allergic subjects. The IgE-binding capacity of natural and recombinant allergen was determined using EAST. RESULTS: The nucleic acid sequence as well as the deduced amino acid sequence consisting of 394 amino acids indicated homology with pollen specific polygalacturonases. Four potential sites for glycosylation and 16 cysteine residues were found. The recombinant expression product exhibited the same molecular size as the natural allergen and was clearly IgE-reactive. CONCLUSION: The newly characterized allergen Phl p 13, which shows homology with polygalacturonases, is clearly different from the allergen designated as Phl p 4 and therefore the high molecular mass fraction is composed of at least two different allergens. A possible reason why this important allergen has not been detected until now is that Phl p 13 and Phl p 4 are hardly separable by one dimensional SDS-PAGE.     

Interferon-gamma-dependent inhibition of late allergic airway responses and eosinophilia by CD8+ gammadelta T cells

We have previously shown that CD8(+)gammadelta T cells decrease late allergic airway responses, airway eosinophilia, T helper 2 cytokine expression and increase interferon-gamma (IFN-gamma) expression. We hypothesized that the effects of CD8(+)gammadelta T cells were IFN-gamma mediated. Brown Norway rats were sensitized to ovalbumin on day 1. Cervical lymph node CD8(+)gammadelta T cells from sensitized animals were treated with antisense oligodeoxynucleotide (5 micromol/l) to inhibit IFN-gamma synthesis or control oligodeoxynucleotide and 3.5 x 10(4) CD8(+)gammadelta T cells were injected intraperitoneally into sensitized recipients on day 13. Rats were challenged with aerosolized ovalbumin on day 15 and lung resistance was monitored over an 8 hr period, after which bronchoalveolar lavage was performed. Control oligodeoxynucleotide treated gammadelta T cells decreased late airway responses and eosinophilia in bronchoalveolar lavage. There was a complete recovery of late airway responses and a partial recovery of airway eosinophilia in recipients of antisense oligodeoxynucleotide treated cells. Macrophage ingestion of eosinophils was frequent in rats administered gammadeltaT cells but reduced in recipients of antisense oligodeoxynucleotide treated cells. These results indicate that CD8(+)gammadelta T cells inhibit late airway responses and airway eosinophilia through the secretion of IFN-gamma. Defective or altered gammadelta T-cell function may account for some forms of allergic asthma.     

Forkhead box P3+ T cells express interleukin-17 in nasal mucosa of patients with both allergic rhinitis and polyposis

The pathogenesis of nasal polyposis remains unclear; it severely affects patients' quality of life and complicates inflammation in adjacent organs such as sinusitis and asthma. Aberrant immune regulatory function in these patients is proposed. The present study aims to examine the regulatory T cells (T(reg) ) in nasal mucosa of patients with allergic rhinitis (AR) and nasal polyposis (NP). Patients with AR or AR/NP were treated with inferior turbinectomy for their inferior turbinate hyperplasia. Surgically removed nasal mucosa was collected to examine the T(reg) by immunohistochemistry and flow cytometry. The results showed that more forkhead box P3 (FoxP3)(+) cells were found in AR with polyps than in those with AR alone. Further studies revealed that these FoxP3(+) T cells from AR/NP group also expressed interleukin (IL)-17. In vitro study showed that staphylococcal enterotoxin B (SEB) induced CD4(+) FoxP3(+) T cells to become FoxP3(+) IL-17(+) cells via facilitating the expression of IL-6, that in synergy with transforming growth factor-beta, induce the expression of IL-17 in FoxP3(+) cells. We conclude that FoxP3(+) IL-17(+) T cells were localized in the nasal mucosa of patients with AR and NP. SEB may play a role in converting FoxP3(+) T(reg) to FoxP3(+) IL-17(+) T cells. The presence of IL-17(+) FoxP3(+) T cells may play a role in the remodelling of the nasal airways in certain people who develop polyps, irrespective of whether or not they are atopic.     

Roles of follicular helper and regulatory T cells in allergic diseases and allergen immunotherapy

Allergic diseases are characterized by overactive type 2 immune responses to allergens and immunoglobulin E (IgE)-mediated hypersensitivity. Emerging evidence suggests that follicular helper T (T_FH) cells, rather than type 2 T-helper (T_H2) cells, play a crucial role in controlling IgE production. However, follicular regulatory T (T_FR) cells, a specialized subset of regulatory T (T_REG) cells resident in B-cell follicles, restricts T_FH cell-mediated help in extrafollicular antibody production, germinal center (GC) formation, immunoglobulin affinity maturation, and long-lived, high-affinity plasma and memory B-cell differentiation. In mouse models of allergic asthma and food allergy, CXCR5⁺ T_FH cells, not CXCR5⁻ conventional T_H2 cells, are needed to support IgE production, otherwise exacerbated by CXCR5⁺ T_FR cell deletion. Upregulation of T_FH cell activities, including a skewing toward type 2 T_FH (T_FH2) and IL-13 producing T_FH (T_FH13) phenotypes, and defects in T_FR cells have been identified in patients with allergic diseases. Allergen immunotherapy (AIT) reinstates the balance between T_FH and T_FR cells in patients with allergic diseases, resulting in clinical benefits. Collectively, further understanding of T_FH and T_FR cells and their role in the immunopathogenesis of allergic diseases creates opportunities to develop novel therapeutic approaches.     

Normative data for total serum immunoglobulin E measurements in children of three ethnicities

BACKGROUND: It is about 20 years since IgE measurements were published for children without atopic disease. It is possible that the recent increase in atopic disease is reflected in altered measurements in subjects who have no clinical expression of atopy. If the measurement of IgE is to be used as a marker for atopy to characterize disease categories, contemporary normative data must be available. OBJECTIVE: To measure total serum IgE in healthy children of three ethnicities born and living in an inner city environment. METHODS: Subjects were aged 1 to < or = 12 years, of Afro-Caribbean, Bangladeshi and white British ethnicities, with no personal history of current atopic disease. An extra 1 mL of blood for the measurement of total serum IgE was collected when blood was taken for other purposes or when a surgical procedure was being undertaken. RESULTS: Measurements were taken from 151 boys (median age 5.4 years) and 106 girls (median age 6.0 years), who included 127 Bangladeshis, 58 Afro-Caribbeans and 72 white British children. Measurements increased with age but were not related to gender or ethnicity. The data were significantly higher than previous measurements by sixfold. CONCLUSION: These contemporary normative data allow the generation of z scores for total IgE measurements for clinical or epidemiological use.     

Bi-directional modulation of T cell-dependent antibody production by prostaglandin E(2).

T cell-dependent Ig production involves interaction between T cells and B cells. This study evaluated the effects of prostaglandin (PG) E(2) on Ig production in a system in which B cells were co-cultured with autologous CD4(+) T cell clones non-specifically activated by anti-CD3. The effects of PGE(2) on T cell-dependent Ig production differed substantially, depending on the T cells employed. We selected six T cell clones that were able to enhance Ig production (resistant T cell clones) and six T cell clones that inhibited Ig production in the presence of PGE(2) (sensitive T cell clones) for comparison. The resistant T cells produced high levels (>1000 pg/ml) of IL-2 and/or IL-4, and expressed high CD40L, OX40 and CD45RA, and low CD45RO. In contrast, sensitive T cells secreted low IL-2 (<500 pg/ml) and IL-4 (<200 pg/ml), and expressed low CD40, OX40 and CD45RA, and high CD45RO. Adding supernatant derived from resistant T cell clones restored Ig production inhibited by PGE(2), while removing IL-2, IL-4 or IL-10 using specific antibodies inhibited Ig production. In addition, we demonstrated a direct effect of PGE(2) on B cells to enhance Ig production. Consistently, in the presence of resistant T cells, PGE(2) increased B cell proliferation and differentiation. In conclusion, the effects of PGE(2) on Ig production consist of its indirect effects through T cells and its direct effects on B cells. The outcome of the effects can be up-regulatory or down-regulatory, depending whether resistant or sensitive T cells are involved.     

Quantitative and functional characteristics of intestinal-homing memory T cells: analysis of Crohn's disease patients and healthy controls

Circulating memory T cells can be subdivided on the basis of beta7 integrin expression. The beta7+ population contains cells primed in the intestine capable of homing back to the gut. We hypothesized that cytokine production by beta7+ memory T cells reflects the specialized mucosal compartment in which they were primed. Flow cytometry of whole blood was used to assess numbers of beta7+ (beta7hi and beta7int) and beta7- memory T cells and their production of Th1 and regulatory cytokines in healthy controls and Crohn's disease patients. In controls, beta7+ and beta7- memory T cells displayed a similar qualitative profile of cytokine production but the beta7+ population was enriched for cytokine-producing effector cells. In addition, the beta7hi population contained more cytokine-producing cells than the beta7int population, suggesting a gradient of cytokine production based on beta7 integrin expression. In active Crohn's disease, there was altered expression of beta7 integrin with a decrease in intestinal-homing memory T cells and an increase in systemic memory T cells. Furthermore, there was a selective loss of IL-10 and increase in TGF-beta in both beta7+ and beta7- memory T cell subsets which may contribute to the pathogenesis of the inflammatory process in Crohn's disease.     

IL-4 is more effective than IL-13 for in vitro differentiation of dendritic cells from peripheral blood mononuclear cells

Dendritic cells (DCs) are the most potent professional antigen-presenting cells which can activate T cells to induce the primary immune response. For clinical studies, DCs are often differentiated in vitro from peripheral blood mononuclear cells (PBMCs) through treatment with granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-4. However, IL-13, a cytokine closely related to IL-4, has also been reported to induce differentiation equally or more efficiently when used with GM-CSF. For the present study, we compared the DC characteristics exhibited by iDCs and LPS-matured DCs differentiated from PBMCs using GM-CSF and IL-4 or IL-13. Physical characteristics examined include cellular morphology and surface phenotype. Functional traits investigated include FITC-dextran uptake, IL-10 and IL-12 production, allostimulation and cytokine production by stimulated T cells and antigen-specific T cell stimulation. Compared with IL-13-derived DCs, IL-4 treatment yielded more differentiated DCs, with extensive dendrites and higher expression of DC-SIGN, DEC-205, CD86 and HLA-DR. In addition, IL-4 DCs were more efficient at inducing allogeneic T cell proliferation and immature IL-4 DCs had higher endocytic activity at low FITC-dextran concentrations (1 microg ml(-1)). Although IL-13 was capable of generating DCs from PBMCs, it was not as effective as IL-4 in generating DC phenotype and functionality. Thus, the use of GM-CSF and IL-4 is the more efficient treatment for inducing DC differentiation from PBMCs.     

Long-term effects of corticosteroid nasal spray on nasal inflammatory cells in patients with perennial allergic rhinitis

BACKGROUND: The effect of long-term topical nasal corticosteroid therapy on nasal inflammatory cells is unclear. OBJECTIVES: To investigate the long-term effect of fluticasone propionate aqueous nasal spray (FPANS) on nasal mucosal inflammatory cells and efficacy in a 1-year study in patients with perennial allergic rhinitis. METHODS: In a 1-year, double-blind, placebo-controlled study of duration we investigated the influence of a topical corticosteroid (FPANS), on Langerhans' cells (CD1a+ cells), T cells, mast cells, eosinophils and macrophages in nasal mucosa in 42 patients with perennial allergic rhinitis. Efficacy was evaluated by nasal symptom score. RESULTS: The FPANS group experienced significantly less sneezing and nasal itching compared with the placebo group. The total symptom score in the FPANS group declined significantly in comparison with baseline (P = 0.007) and placebo group (P = 0.009). After 1 year of active treatment, a significant decrease was seen in the epithelium in numbers of Langerhans' cells, CD3+, CD4+, CD8+ cells, mast cells and eosinophils. In the lamina propria, there was a significant decrease in eosinophils. CONCLUSION: These findings show that FPANS treatment results in a decrease of nasal inflammatory cells. Furthermore, the efficacy of FPANS improves after prolonged treatment.     

Gluten activation of peripheral blood T cells induces a Th0-like cytokine pattern in both coeliac patients and controls

Coeliac disease is apparently a T cell-mediated disease, precipitated in the proximal small intestine of susceptible individuals by gluten. Preferential presentation of gluten peptides most probably takes place in coeliac mucosa by the disease-associated HLA-DQ2 and -DQ8 molecules. In peripheral blood, however, both HLA-DR, -DQ and -DP-restricted T cell responses to gluten have been observed. We examined gluten-specific T cell clones (TCC) derived from peripheral blood for cytokine production to see if their profiles were related to the HLA restriction or the disease state of the donors. As previously found for mucosal TCC, the main product was interferon-gamma (IFN-γ), often with additional IL-4, IL-5, IL-6, IL-10, tumour necrosis factor, and transforming growth factor-beta. Regardless of restriction element or disease state, gluten-reactive TCC from peripheral blood therefore seem to secrete cytokines compatible with a Th0 profile.     

Nasal carriage of Staphylococcus aureus in house dust mite allergic patients and healthy controls

BACKGROUND: Staphylococcal colonization may influence the course of allergic diseases such as atopic dermatitis or allergic rhinitis. The frequency of Staphylococcus aureus (SA) nasal carriage and its possible influence on persistent allergic rhinitis was investigated. METHODS: In nasal lavages from 22 patients with house dust mite allergy and 18 healthy controls, the number of SA colony forming units per ml were assessed and related to nasal symptom scores, the concentrations of three inflammatory cell activation markers, nasal total IgE and 17 cytokines in nasal secretions. RESULTS: SA was found in 15/22 allergic patients and 4/18 controls (P < 0.01). Comparing allergic SA carriers with allergic noncarriers, nasal symptom scores tended to be higher (P < 0.1), and the cell activation markers ECP (10(2.23+/-0.33)vs 10(1.45+/-0.50) ng/ml; P < 0.05) and elastase (10(2.70+/-0.21)vs 10(2.12+/-0.34) ng/ml; P < 0.01), and nasal total IgE-levels (10(1.66+/-0.38)vs 10(1.2+/-0.28) kU/ml; P < 0.05) were significantly higher in allergic SA carriers. Nasal SA carriers had a higher nasal IL-13/IFN-gamma ratio (P < 0.01), and this was correlated with higher nasal total IgE in allergic patients (r = 0.6, P < 0.05). CONCLUSION: Nasal SA carriage is frequent in patients with persistent allergic rhinitis. The data of this study suggest that they are not only secondary bystanders, but actively modulate the disease by promoting local IgE production.     

Multispecific and heterogeneous recognition of the gag protein by cytotoxic T lymphocytes (CTL) from HIV-infected patients: factors other than the MHC control the epitopic specificities.

The HIV gag polyprotein is a major target for recognition by CTL in infected humans. Using recombinant vaccinia viruses (rVV) expressing truncations of the p24gag, and the p18gag, p15gag and HIV-2 p56gag proteins, the characterization of epitope regions recognized by in vitro-stimulated peripheral blood mononuclear cells (PBMC) from 18 infected patients has been studied. The gag-specific response of most individuals is polyclonal and multispecific, and interindividual variations between target epitope regions were frequently observed, despite shared MHC alleles. As CTL may play an important role in the control of HIV replication in infected hosts, these results have important implications for designing vaccine strategies.     

Lenalidomide enhancement of human T cell functions in human immunodeficiency virus (HIV)-infected and HIV-negative CD4 T lymphocytopenic patients

Suppressed T cell functions in human immunodeficiency virus (HIV) infection were identified and corrected by lenalidomide in middle-aged HIV-infected patients. Chemotaxis of T cells from HIV-infected men (n = 6, mean 43 years) to sphingosine 1-phosphate (S1P) and CCL21 was significantly lower than that of HIV-negative men (n = 6, mean 41 years), and was enhanced significantly up to control levels by 100 and 1000 nM lenalidomide. Generation of interleukin (IL)-2, but not interferon (IFN)-γ, by T cells of middle-aged HIV-infected men was significantly lower than that for controls and was increased significantly by 10-1000 nM lenalidomide up to a maximum of more than 300%. CD4 and CD8 T cells isolated from healthy middle-aged men and reconstituted in vitro at a low CD4 : CD8 ratio typical of HIV infection had depressed chemotaxis to S1P, but not CCL21, and generation of IL-2, but not IFN-γ. Significant enhancement of chemotaxis to S1P and CCL21 was induced by 100-1000 nM lenalidomide only for normal T cells at a low CD4 : CD8 ratio. T cells from HIV-negative middle-aged CD4 T lymphocytopenic patients (n = 3), with a CD4 : CD8 ratio as low as that of HIV-infected patients, had similarly diminished chemotaxis to S1P and CCL21, and depressed generation of IL-2, but not IFN-γ. Lenalidomide at 30-1000 nM significantly enhanced chemotaxis to S1P and IL-2 generation for T cells from HIV-negative CD4 T lymphocytopenic patients as from HIV-infected patients, with less effect on CCL21-elicited chemotaxis and none for IFN-γ generation. Defects in functions of T cells from middle-aged HIV-infected men are partially attributable to CD4 T lymphocytopenia and are corrected by lenalidomide.