Mikila R. Jacobson

Grass Pollen Immunotherapy Induces Mucosal and Peripheral IL-10 Responses and Blocking IgG Activity

T regulatory cells and IL-10 have been implicated in the mechanism of immunotherapy in patients with systemic anaphylaxis following bee stings. We studied the role of IL-10 in the induction of clinical, cellular, and humoral tolerance during immunotherapy for local mucosal allergy in subjects with seasonal pollinosis. Local and systemic IL-10 responses and serum Ab concentrations were measured before/after a double-blind trial of grass pollen (Phleum pratense, Phl P) immunotherapy. We observed local increases in IL-10 mRNA-positive cells in the nasal mucosa after 2 years of immunotherapy, but only during the pollen season. IL-10 protein-positive cells were also increased and correlated with IL-10 mRNA+ cells. These changes were not observed in placebo-treated subjects or in healthy controls. Fifteen and 35% of IL-10 mRNA signals were colocalized to CD3+ T cells and CD68+ macrophages, respectively, whereas only 1–2% of total CD3+ cells and 4% of macrophages expressed IL-10. Following immunotherapy, peripheral T cells cultured in the presence of grass pollen extract also produced IL-10. Immunotherapy resulted in blunting of seasonal increases in serum allergen Phl p 5-specific IgE, 60- to 80-fold increases in Phl p 5-specific IgG, and 100-fold increases in Phl p 5-specific IgG4. Post-immunotherapy serum exhibited inhibitory activity, which coeluted with IgG4, and blocked IgE-facilitated binding of allergen-IgE complexes to B cells. Both the increases in IgG and the IgG “blocking” activity correlated with the patients’ overall assessment of improvement. Thus, grass pollen immunotherapy may induce allergen-specific, IL-10-dependent “protective” IgG4 responses.

Grass pollen immunotherapy induces Foxp3-expressing CD4+CD25+ cells in the nasal mucosa

BACKGROUND Regulatory T (Treg) cells play an important role in controlling allergic inflammation. The transcription factor Foxp3 regulates the development and function of natural and adaptive CD4(+)CD25(+) Treg cells. OBJECTIVES We sought to examine the effect of grass pollen injection immunotherapy on the numbers of Foxp3(+)CD4(+) and Foxp3(+)CD25(+) T cells in and out of season and their expression of IL-10 in the nasal mucosa of patients with hay fever. METHODS Nasal biopsy specimens were obtained from untreated patients with hay fever, participants with grass pollen allergy who had received 2 years of immunotherapy, and healthy control subjects. Dual-immunofluorescence microscopy was used to enumerate and colocalize Foxp3 expression to CD4(+) and CD25(+) T cells in the nasal mucosa. Triple staining was performed to colocalize Foxp3(+) cells to CD3(+)CD25(+) and CD3(+) IL-10-expressing cells. RESULTS At peak season, numbers of Foxp3(+)CD25(+) (P = .02) and Foxp3(+)CD4(+) (P = .03) cells were significantly increased in the nasal mucosa of immunotherapy-treated patients compared with numbers before treatment. Foxp3(+)CD25(+) (P = .03) and Foxp3(+)CD4(+) (P = .04) cells were also greater in immunotherapy-treated patients out of season compared with those in untreated patients with hay fever. Within the immunotherapy-treated group, 20% of CD3(+)CD25(+) cells expressed Foxp3, and 18% of Foxp3(+)CD3(+) cells were IL-10 positive. CONCLUSION The presence of local Foxp3(+)CD25(+)CD3(+) cells in the nasal mucosa, their increased numbers after immunotherapy, and their association with clinical efficacy and suppression of seasonal allergic inflammation support a putative role for Treg cells in the induction of allergen-specific tolerance in human subjects.

Long-term tolerance after allergen immunotherapy is accompanied by selective persistence of blocking antibodies

BACKGROUND Grass pollen immunotherapy for allergic rhinitis is a disease-modifying treatment that results in long-term clinical tolerance lasting years after treatment discontinuation. Active treatment is associated with generation of inhibitory grass pollen-specific IgG antibodies capable of blocking allergen-IgE interactions. OBJECTIVES We sought to investigate the involvement of IgG-associated inhibitory antibodies with long-term clinical tolerance after discontinuation of grass pollen immunotherapy. METHODS We conducted a 4-year study in which patients who had moderate-to-severe allergic rhinitis underwent a randomized, double-blind, placebo-controlled discontinuation of subcutaneous grass pollen immunotherapy. All subjects received grass pollen immunotherapy injections for 2 years (n = 13), followed by a further 2 years of either active (n = 7) or placebo (n = 6) injections. Clinical outcomes included seasonal symptoms and use of rescue medication. Serum specimens were collected at baseline and after 2 and 4 years for quantification of allergen-specific IgG antibodies. Sera were also tested for IgG-dependent inhibitory bioactivity against IgE-allergen binding in cellular assays by using flow cytometry and confocal microscopy to detect binding of IgE-grass pollen allergen complexes to B cells. RESULTS Clinical improvement was maintained after 2 years of discontinuation. Although immunotherapy-induced grass pollen-specific IgG1 and IgG4 levels decreased to near-preimmunotherapy levels during discontinuation, inhibitory bioactivity of allergen-specific IgG antibodies was maintained unchanged. CONCLUSION Grass pollen immunotherapy induces a subpopulation of allergen-specific IgG antibodies with potent inhibitory activity against IgE that persists after treatment discontinuation and that could account for long-term clinical tolerance.

Sublingual grass pollen immunotherapy is associated with increases in sublingual Foxp3-expressing cells and elevated allergen-specific immunoglobulin G4, immunoglobulin A and serum inhibitory activity for immunoglobulin E-facilitated allergen binding to B cells

Background The mechanisms of sublingual immunotherapy (SLIT) are less well understood than those of subcutaneous immunotherapy (SCIT).

Grass Pollen Immunotherapy Induces an Allergen-Specific IgA2 Antibody Response Associated with Mucosal TGF-β Expression

Allergen immunotherapy (IT) has long-term efficacy in IgE-mediated allergic rhinitis and asthma. IT has been shown to modify lymphocyte responses to allergen, inducing IL-10 production and IgG Abs. In contrast, a putative role for IgA and local TGF-β-producing cells remains to be determined. In 44 patients with seasonal rhinitis/asthma, serum IgA1, IgA2, and polymeric (J chain-containing) Abs to the major allergen Phl p 5 were determined by ELISA before and after a 2-year double-blind trial of grass pollen (Phleum pratense) injection IT. Nasal TGF-β expression was assessed by in situ hybridization. Sera from five IT patients were fractionated for functional analysis of the effects of IgA and IgG Abs on IL-10 production by blood monocytes and allergen-IgE binding to B cells. Serum Phl p 5-specific IgA2 Abs increased after a 2-year treatment (∼8-fold increase, p = 0.002) in contrast to IgA1. Increases in polymeric Abs to Phl p 5 (∼2-fold increase, p = 0.02) and in nasal TGF-β mRNA (p = 0.05) were also observed, and TGF-β mRNA correlated with serum Phl p 5 IgA2 (r = 0.61, p = 0.009). Post-IT IgA fractions triggered IL-10 secretion by monocytes while not inhibiting allergen-IgE binding to B cells as observed with IgG fractions. This study shows for the first time that the IgA response to IT is selective for IgA2, correlates with increased local TGF-β expression, and induces monocyte IL-10 expression, suggesting that IgA Abs could thereby contribute to the tolerance developed in IT-treated allergic patients.

Grass pollen immunotherapy : efficacy and safety during a 4-year follow-up study

Grass pollen immunotherapy is effective, although efficacy must be balanced against side‐effects. In a double‐blind, placebo‐controlled trial of 40 adult patients with summer hay fever, immunotherapy with a depot grass pollen extract (Phleum pratense, Alutard SQ) reduced symptoms and medication requirements with an acceptable minimal level of side‐effects (31). The original placebo group, as well as the actively treated group, have now received active immunotherapy in an open fashion for a further 3 years. An important question was whether continued injection treatment was accompanied by maintained clinical improvement. By analysis of diary symptoms, rescue medication, and visual analogue scores during the pollen season, we show that efficacy was maintained throughout the 3–4‐year study period. Clinical improvement was accompanied by a sustained and marked decrease in immediate conjunctival allergen sensitivity and a further significant decrease in the size of the allergen‐induced late cutaneous response. In contrast, an initial decrease in the allergen‐induced immediate cutaneous response was not maintained at 3–4 years. Of the patients, 37/40 completed the first year, 33/40 the second year, and 32/40 the third year of treatment. Patients dropped out for reasons other than the outcome of immunotherapy. During a total of 2598 injections, five immediate systemic reactions were observed, all during the induction (not maintenance) phase, and all occurred within 10 min of injection and responded promptly to adrenaline. Grass pollen immunotherapy is effective and safe, provided it is performed on carefully selected patients by trained physicians with immediate access to resuscitative measures.

Influence of prolonged treatment with topical corticosteroid (fluticasone propionate) on early and late phase nasal responses and cellular infiltration in the nasal mucosa after allergen challenge

We have examined the effect of prolonged treatment with topical corticosteroid on allergen‐induced early and late nasal responses and the associated inflammatory cell infiltrate in grass pollen sensitive allergic rhinitics. Following a randomized double‐blind 6 week treatment period with fluticasone propionate 200 μg aqueous nasal spray twice daily or matched placebo spray, nasal provocation was performed using Timothy grass pollen extract. Nasal symptoms were recorded at intervals from 0 to 24 h. Nasal biopsies were performed before treatment and at 24 h after allergen and processed for immunohistology. When corticosteroid‐treated patients were compared with the placebo group there was an approximately 50% decrease in the size of the early (0‐60 min) response and almost complete inhibition of late (1–24 h) nasal symptoms after allergen challenge. After allergen challenge markedly fewer T lymphocytes and CD25+ (interleukin‐2 receptor bearing) cells were observed in both the epithelium and submucosa in fluticasone treated patients compared with the placebo group. Significantly less total and activated eosinophils were observed, particularly within the nasal epithelium. Submucosal mast cell counts were decreased, whereas increased numbers of submucosal neutrophils were observed. These results confirm that topical corticosteroid treatment inhibits allergen‐induced early and late nasal responses. This may possibly occur following a decrease in T lymphocytes and/or mast cells and their products and a consequent reduction in tissue eosinophilia.

Effect of cetirizine and prednisolone on cellular infiltration and cytokine mRNA expression during allergen‐induced late cutaneous responses

Background The ability of cetirizine to inhibit eosinophil infiltration into the sites of allergen‐induced cutaneous late‐phase reactions is controversial. A previous skin biopsy study gave negative results with 15 mg of cetirizine as a single dose.

Evidence for Th2-type T helper cell control of allergic disease in vivo.

Atopic allergic asthma is characterized by the synthesis of specific IgE antibody directed against allergens. This may lead to acute bronchoconstriction and bronchial mucosal edema through the actions of mast cell and basophil-derived mediators triggered via high affinity IgE receptors [29]. It is now clear that a cellular inflammatory infiltrate is also a feature of the asthmatic bronchial mucosa even in very mild asymptomatic disease [14, 32]. Increased eosinophil numbers are detected in bronchoalveolar lavage and bronchial biopsy specimens from asthmatic subjects when compared to controls [5, 34, 66] and the detection of eosinophil granule proteins in bronchoaveolar lavage (BAL) fluid and lung tissue also supports a role for these cells [17, 66]. Eosinophil granule proteins damage respiratory epithelium in vitro, and together with release of lipid mediators, this may contribute to bronchial hyperresponsiveness in vivo in asthma [23]. In recent years the pivotal role of T lymphocytes in control of IgE synthesis has been defined in vitro [62]. The balance of T cell-derived cytokines interleukin4 (IL-4) and interferon-3, (IFN-3,) is important in initial isotype switching, and a second signal which may depend on direct T cell contact is required [11, 28, 58]. T lymphocytes are also implicated in the control of eosinophil development and activation [2]. In particular the cytokine IL-5 appears to be specific in promoting development [8, 13], adhesion [65] and activation [36] of eosinophils rather than neutrophils. IL-3 and granulocyte/macrophage-colony-stimulating factor (GM-CSF) are also active in eosinophil development and activation [43, 54]. The subdivision of murine T helper (Th) cell clones on the basis of cytokine profile [41] lead to the concept that the pattern of cytokines released during T cell-mediated immune responses might determine the nature of the resulting inflammatory processes [40]. In particular the Th2 pattern with IL-4 unopposed by IFN-3,, together with IL-5, IL-3 and GM-CSF might be expected to predominate in allergic diseases which are characterized by IgE synthesis and eosinophilic inflammation [39]. Although initial screening of human T cell clones did not

CC Chemokine Receptor 4 (CCR4) in human allergen-induced late nasal responses

To cite this article: Banfield G, Watanabe H, Scadding G, Jacobson MR, Till SJ, Hall DA, Robinson DS, Lloyd CM, Nouri‐Aria KT, Durham SR. CC Chemokine Receptor 4 (CCR4) in human allergen‐induced late nasal responses. Allergy 2010; 65: 1126–1133.