Allergen extract-induced interleukin-10 in human memory B cells inhibits immunoglobulin E production

Background Elevated specific IgE antibody levels are common in atopic individuals, caused by T-helper type 2-dominated B cell activation. The induction of antigen-specific IL-10 secreting T cells is discussed as an important mechanism during specific immunotherapy. By contrast the presence and function of B cell-derived IL-10 is not well defined yet.
Objective We investigated whether type-I allergen extracts induce IL-10 expression in human B cells and analysed its functional role on IgE production.
Methods Human peripheral B cells were stimulated with grass pollen, house dust mite (HDM) ( Dermatophagoides pteronyssimus ; Der p) and dog allergen extract. Expression of IL-10 by activated human B cells was determined by flow cytometric analysis and ELISA. Functional analysis considering immunoglobulin production was assayed by ELISA.
Results The allergen extracts studied induced IL-10 expression in B cells. However, the ability to induce IL-10 differed between the allergen extracts. The most potent allergen extract was dog (169±28 pg/mL), followed by grass pollen (141±10 pg/mL) and HDM allergen (125±11 pg/mL). Upon allergen extract stimulation only CD27 expressing memory B cells produced IL-10 and co-expressed the very early activation antigen CD69. The addition of allergen extracts to B cells activated by anti-CD40 and IL-4 selectively inhibited IgE which was dependent on allergen extract-induced IL-10. By contrast the other immunoglobulin subclasses like IgA, IgG or IgM were not altered upon allergen extract challenge.
Conclusion Our data indicate that allergen-activated memory B cells can modulate IgE production through secretion of IL-10.     

Immune regulation by CD4+CD25+ T cells and interleukin-10 in birch pollen-allergic patients and non-allergic controls

BACKGROUND: CD4+CD25+ regulatory T (Treg) cells and the cytokines IL-10 or TGF-beta play key roles in the maintenance of T cell homeostasis and tolerance to infectious and non-infectious antigens such as allergens. OBJECTIVE: To investigate the regulation of immune responses to birch pollen allergen compared with influenza antigen by Treg cells obtained from birch pollen-allergic patients and non-allergic controls. METHODS: Peripheral blood was collected from 10 birch pollen-allergic patients and 10 non-allergic healthy controls. CD4+CD25+ and CD4+CD25- cells isolated by magnetic-activated cell sorting were co-cultured and stimulated with birch pollen extract or influenza vaccine in the absence or presence of anti-IL-10 or soluble TGF-betaRII. RESULTS: CD4+CD25+ cells from non-allergic controls were able to suppress influenza antigen and birch pollen stimulated effector cell proliferation, whereas CD4+CD25+ cells from allergic patients suppressed influenza antigen-, but not birch pollen-stimulated proliferation. The production of Th1 cytokines, but not Th2 cytokines, was suppressed by CD4+CD25+ cells from both allergic patients and controls, upon stimulation with birch pollen extract. Neutralization of IL-10 led to significantly increased production of IFN-gamma in cultures with CD4+CD25- T effector cells. In addition, six-fold higher concentrations of TNF-alpha were detected after neutralization of IL-10 in both CD4+CD25- and CD4+CD25+ cell cultures from allergic patients and controls. CONCLUSION: We demonstrate that the allergen-specific suppressive function of CD4+CD25+ cells from allergic patients is impaired compared with non-allergic controls. Moreover, neutralization of IL-10 enhances the production of TNF-alpha, suggesting counter-acting properties of IL-10 and TNF-alpha, where IL-10 promotes tolerance and suppression by Treg cells and TNF-alpha promotes inflammatory responses.     

Human CD4+CD25+ T cells derived from the majority of atopic donors are able to suppress TH1 and TH2 cytokine production

BACKGROUND: Recently, it has been established that CD4(+)CD25(+) T cells with regulatory capacity are present in human peripheral blood, inhibiting allogeneic proliferation and cytokine production of preactivated CD4(+)CD25(-) respond-er T cells. OBJECTIVE: The aim of this study was to analyze in an allergen-specific setting whether such regulatory CD4(+)CD25(+) T cells also exist and function normally in atopic individuals, especially concerning the inhibition of T(H)2 cytokines. METHODS: For this purpose, CD4(+)CD25(-) or CD4(+)CD25(+) T cells from donors allergic to grass or birch pollen (mainly with rhinitis) or from healthy nonatopic donors were stimulated in the presence of autologous, mature, monocyte-derived, allergen-pulsed dendritic cells, and the preactivated CD4(+)CD25(+) T cells were added to CD4(+)CD25(-) T cells during restimulation. RESULTS: CD4(+)CD25(+) T cells from the nonatopic donors and from the majority of the patients investigated proliferated poorly, produced fewer cytokines, and inhibited the proliferation and T(H)1 (IFN-gamma) and T(H)2 (IL-4 and IL-5) cytokine production of CD4(+)CD25(-) T cells but not IL-10 production. The suppression of CD4(+)CD25(-) T cells by CD4(+)CD25(+) T cells was at least partially antigen unspecific and not reversible with anti-IL-10, anti-transforming growth factor beta, or anti-cytotoxic T lymphocyte-associated antigen 4 mAb but was reversible with IL-2. In some atopic patients preactivated CD4(+)CD25(+) T cells reproducibly showed strong proliferative responses, produced higher amounts of IL-4 and IL-10 than CD4(+)CD25(-) T cells, and suppressed only the IFN-gamma production of CD4(+)CD25(-) T cells. CONCLUSION: These data indicate that regulatory CD4(+)CD25(+) T cells are present and functional in most atopic patients with allergic rhinitis and are able to inhibit T(H)1, as well as T(H)2, cytokine production.     

Evaluation of CD4+ T cells proliferating to grass pollen in seasonal allergic subjects by flow cytometry

Our objective was to characterize T-cell responses to Phleum pratense in grass pollen allergic individuals and healthy controls using the fluorescent dye PKH26. Peripheral blood mononuclear cells were stimulated with P. pratense, or with recall antigens, and CD3+/CD4+ and CD3+/CD8+ T-cells that had proliferated were analysed by flow cytometry. In the presence of P. pratense CD4+/CD3+ T-cells proliferated more in grass pollen sensitive atopic patients than in nonallergic controls or in nongrass pollen sensitive atopic subjects. PPD and TT recall antigens elicited uniformly high proliferation in all T-cell subsets. Only half of pollen sensitive patients also had an increased proliferation of CD3+/CD8+ T-cells in response to P. pratense. We determined precursor frequency of CD4+ T cells in the original population that responded to P. pratense and found values ranging from 1 x 10-3 to 0.6 x 10-1, in the same range as those measured for PPD and TT. In conclusion, grass pollen sensitive atopic patients show enhanced CD4+ T-cell reactivity to P. pratense, and this could be related to the presence of elevated numbers of circulating allergen-specific CD4+ T cells. This flow cytometric method should allow the identification of other phenotypic markers such as intracellular cytokines in allergen specific responding CD4+ T cells.     

Depletion of CD4+CD25+CD127lo regulatory T cells does not increase allergen‐driven T cell activation

Background It has been suggested that allergic diseases are caused by defective suppression of allergen‐specific Th2 cells by CD4⁺CD25⁺ regulatory T cells. However, such studies have been hampered by the difficulty in distinguishing regulatory T cells from CD25‐expressing activated T cells. Recently, it was shown that conventional T cells expressed high levels of CD127, whereas regulatory T cells were CD127^lo, allowing discrimination between these distinct T cell subpopulations. Objective The aim of this study was to study whether the putative regulatory subset defined as CD4⁺CD25⁺CD127^lo was involved in grass pollen‐reactive T cell responses. Methods Peripheral blood mononuclear cells (PBMCs) were obtained from allergic donors and non‐atopic controls out of season. Grass pollen‐induced cytokine production and proliferation were compared in cultures of undepleted cells and cells depleted of CD4⁺CD25⁺, CD4⁺CD25⁺CD127^hi or CD4⁺CD25⁺CD127^lo T cells. Results Undepleted cell cultures from allergic patients showed significantly increased proliferation and Th2 cytokine production compared with non‐atopic controls. Depletion of all CD25⁺ T cells did not increase cytokine production or proliferation, and more importantly, no increase in Th2 cytokine production or proliferation was observed in cell cultures depleted of CD4⁺CD25⁺CD127^lo cells (putative regulatory T cells) compared with undepleted PBMCs in both the allergic and the non‐atopic group. Conclusion Our study showed that T cells from grass pollen‐allergic patients and non‐atopic controls responded very differently to grass pollen extract, but this difference could not be explained by differences in regulatory T cell function. Further studies are needed to understand the importance of regulatory T cells in allergy.     

T lymphocytes expressing CCR3 are increased in allergic rhinitis compared with non-allergic controls and following allergen immunotherapy

BACKGROUND: In T cell-associated allergic inflammation, homing of T-helper 2 (Th2) effector cells to mucosal sites may be influenced by chemokine receptor expression. Previous studies have identified CCR3 and CCR4 as putative markers of Th2 cells and CCR5 and CXCR3 as markers of Th1 cells. The aim of this study was to assess differential chemokine receptor expression from symptomatic atopic grass pollen-sensitive subjects, compared with patients on high-dose allergen injection immunotherapy (IT) and healthy controls. METHODS: We examined chemokine receptor expression (CCR1-7 and CXCR1-4) by flow cytometry of peripheral blood CD4+ and CD8+ T cells. We also depleted peripheral blood mononuclear cell (PBMC) populations of CCR3+ CD4+ cells by magnetic bead separation and cells were stimulated with grass pollen allergen for 6 days. Cytokine production was measured by enzyme-linked immunosorbent assay. RESULTS: On freshly isolated PBMC, atopic individuals exhibited increased numbers of CCR3+ CD4+ cells compared with normal controls (P < 0.01). CCR3 expression in IT patients was reduced compared with matched atopic rhinitic controls (P < 0.05) and comparable with that observed in normal subjects. Depletion of CCR3+ CD4+ cells from allergen-stimulated PBMC cultures resulted in decreased interleukin (IL)-5 production compared with whole CD4+ populations (P < 0.05). Freshly isolated CCR3+ CD4+ cells have significantly higher intracellular IL-4 and lower IFN-gamma levels than CCR3- CD4+ cells. CD4+ T cells cultured from both peripheral cells and nasal biopsies demonstrated increased expression of CCR3 in the presence of IL-4 (P < 0.05). CONCLUSION: CCR3+ CD4+ T cells are increased in allergic rhinitis, are reduced by allergen IT, have a Th2 phenotype and contribute to allergen-specific responses. Strategies against CCR3+ T cells may be effective in human allergic diseases.     

Fluticasone propionate increases CD4CD25 T regulatory cell suppression of allergen-stimulated CD4CD25 T cells by an IL-10-dependent mechanism

BACKGROUND: Corticosteroids are the most effective anti-inflammatory therapy for allergic diseases, and these drugs inhibit TH2 T-cell activation. We previously reported that CD4+CD25+ T cells from atopic donors suppressed allergen-stimulated T cells less than those from nonatopic donors. OBJECTIVE: We sought to determine the effect of fluticasone propionate (FP) on allergen-stimulated CD4+CD25- T cells and on the suppressive ability of CD4+CD25+ T cells. METHODS: CD4+CD25+ and CD4+CD25- T cells were separated from peripheral blood of atopic and nonatopic volunteers and cultured alone or mixed in the presence of allergen. Effects of FP were assessed by means of addition to cultures or preincubation with CD4+CD25+ T cells. RESULTS: FP inhibited allergen-stimulated proliferation of CD4+CD25- T cells in a dose-dependent manner. Preincubation of CD4+CD25+ T cells in FP increased subsequent suppressive activity of these cells in allergen-stimulated cultures with CD4+CD25- T cells. This effect was seen when cells were obtained from both nonatopic and atopic donors but was less for cells obtained from atopic individuals. Prior exposure of CD4+CD25+ T cells to FP also increased subsequent IL-10 production by these cells when stimulated with allergen, and addition of anti-IL-10 antibody reversed the steroid-induced enhancement of suppression in mixed cultures. CONCLUSION: Increased suppression by CD4+CD25+ T cells might play a role in anti-inflammatory effects of corticosteroids in asthma and allergic diseases.     

Defective suppression of Th2 cytokines by CD4+CD25+ regulatory T cells in birch allergics during birch pollen season

BACKGROUND: CD4(+)CD25+ regulatory T cells suppress proliferation and cytokine production by human T cells both to self-antigens and exogenous antigens. Absence of these cells in human newborns leads to multiple autoimmune and inflammatory disorders together with elevated IgE levels. However, their role in human allergic disease is still unclear. OBJECTIVE: This study aimed to evaluate the capacity of CD4(+)CD25+ regulatory T cells to suppress proliferation and cytokine production outside and during birch-pollen season in birch-allergic patients relative to non-allergic controls. METHODS: CD4+ cells were obtained from blood of 13 birch-allergic patients and six non-allergic controls outside pollen season and from 10 birch-allergic patients and 10 non-allergic controls during birch-pollen season. CD25+ and CD25- fractions were purified with magnetic beads and cell fractions, alone or together in various ratios, were cultured with antigen-presenting cells and birch-pollen extract or anti-CD3 antibody. Proliferation and levels of IFN-gamma, IL-13, IL-5 and IL-10 were measured by thymidin incorporation and ELISA, respectively. Numbers of CD25+ cells were analysed by flow cytometry. RESULTS: CD4(+)CD25+ regulatory T cells from both allergics and non-allergics potently suppressed T cell proliferation to birch allergen both outside and during birch-pollen season. However, during season CD4(+)CD25+ regulatory T cells from allergic patients but not from non-allergic controls were defective in down-regulating birch pollen induced IL-13 and IL-5 production, while their capacity to suppress IFN-gamma production was retained. In contrast, outside pollen season the regulatory cells of both allergics and non-allergic controls were able to inhibit T-helper 2 cytokine production. CONCLUSION: This is the first study to show differential suppression of Th1 and Th2 cytokines, with CD4(+)CD25+ regulatory T cells from birch-pollen-allergic patients being unable to down-regulate Th2, but not Th1 responses during birch-pollen season.     

Cat allergen peptide immunotherapy reduces CD4(+) T cell responses to cat allergen but does not alter suppression by CD4(+) CD25(+) T cells: a double-blind placebo-controlled study

BACKGROUND: We have previously described both modification of allergen immunotherapy using peptide fragments, and reduced regulation of allergen stimulated T cells by CD4(+) CD25(+) T cells from allergic donors when compared with nonallergic controls. It has been suggested that allergen immunotherapy induces regulatory T cell activity: we hypothesized that allergen peptide immunotherapy might increase suppressive activity of CD4(+) CD25(+) T cells. OBJECTIVE: To examine cat allergen-stimulated CD4 T cell responses and their suppression by CD4(+) CD25(+) T cells before and after cat allergen peptide immunotherapy in a double-blind placebo-controlled study. METHODS: Peripheral blood was obtained and stored before and after peptide immunotherapy or placebo treatment. CD4(+) and CD4(+) CD25(+) were then isolated by immunomagnetic beads and cultured with allergen in vitro. RESULTS: Comparing cells from blood taken before with that after peptide immunotherapy there was a significant reduction in both proliferation and IL-13 production by allergen-stimulated CD4+ T cells, whereas no change was seen after placebo. CD4(+) CD25(+) T cells suppressed both proliferation and IL-13 production by CD4(+) CD25(-) T cells before and after therapy but peptide therapy was not associated with any change in suppressive activity of these cells. CONCLUSION: Allergen peptide immunotherapy alters T cell response to allergen through mechanisms other than changes in CD4(+) CD25(+) T cell suppression.     

High dose allergen stimulation of T cells from house dust mite-allergic subjects induces expansion of IFN-gamma+ T Cells, apoptosis of CD4+IL-4+ T cells and T cell anergy

BACKGROUND: During clinically effective allergen-specific immunotherapy a shift in cytokine dominance from IL-4, IL-5 predominant to IFN-gamma predominant has been observed. As antigen concentration influences Th cell priming, this study aimed to determine the effect of different allergen concentrations on human house dust mite (HDM)-specific T cell production of IL-4 and IFN-gamma, proliferation and apoptosis. METHODS: HDM-allergic donor PBMC were cultured for 14 days with different concentrations of HDM extract (1, 10 and 100 microg/ml). T cell intracellular IL-4 and IFN-gamma, division (CFSE labelling) and apoptosis (active caspase-3 staining) were analysed by flow cytometry. Proliferation was assessed by (3)H-thymidine incorporation. RESULTS: Increased CD4+IFN-gamma+ and CD8+IFN-gamma+ T cell numbers were observed in high allergen concentration cultures compared with low concentration cultures whereas there were no differences in CD4+IL-4+ and CD8+IL-4+ T cell numbers. CFSE cell labelling revealed that high allergen concentration favours the expansion of IFN-gamma-producing CD4+ T cells. The proportion of apoptotic cells increased with allergen concentration and there was preferential apoptosis of CD4+IL-4+ T cells. HDM-induced proliferation was decreased in high allergen concentration cultures; this was reversible by IL-2 consistent with anergy. CONCLUSION: These results show that T cell division and apoptosis contribute to the cytokine skewing to predominant IFN-gamma production by T cells observed at high allergen concentration. Thus the use of hypoallergenic T cell reactive preparations which can be given safely at higher doses than natural extracts may enhance efficacy of allergen-specific immunotherapy.     

Induction of T ''regulatory'' cells by standardized house dust mite immunotherapy: an increase in CD4+CD25+ interleukin-10+ T cells expressing peripheral tissue trafficking markers

BACKGROUND: Clinically effective subcutaneous allergen-specific immunotherapy (SIT) is associated with altered circulating T cell cytokine production and altered local cytokine responses with increased IL-10 following allergen challenge in target organs. OBJECTIVE: This study aimed to elucidate mechanisms for these T cell changes, by examining surface expression of markers for peripheral tissue trafficking on circulating cytokine-positive T cells following standardized house dust mite- (HDM-) SIT. METHODS: A randomized conventional HDM immunotherapy study was performed on a panel of 12 HDM-allergic subjects. Nine subjects received treatment with conventional HDM immunotherapy using a standardized extract and three subjects were treated by standard pharmacotherapy alone. Symptom and medication scores and allergen-induced cutaneous late-phase responses were assessed before and 9 months after institution of therapy. Before and at 3 and 9 months of SIT, peripheral blood mononuclear cells were cultured for 14 days with HDM extract and CD4+ and CD8+ T cell expression of CD62L, CD49d and CCR5 and production of IL-10, IFN-gamma and IL-4 were analysed by flow cytometry. Allergen-specific T cell proliferation was assessed by 3H-thymidine incorporation. RESULTS: At 9 months, all SIT-treated patients showed reduced symptom scores and late-phase cutaneous responses to HDM compared with baseline levels. The proportions of CD4+ T cells which were IL-10+ were increased (P < 0.01), and the proportions of CD4+ and CD8+ T cells which were IL-4+ decreased (P < 0.05) compared with baseline. CD4+ and CD8+ T cell IFN-gamma production, expression of surface markers for peripheral tissue trafficking and allergen-specific proliferation remained unchanged during SIT treatment. However, increased proportions of CD4+CD62L(-), CD4+CD49d(hi), CD4+CCR5+ T cells expressing IL-10 were detected at 9 months of SIT compared with baseline (P < 0.05). IL-10 staining co-localized with CD4+CD25+ T cells. CONCLUSION: Clinically effective subcutaneous immunotherapy with a standardized HDM Dermatophagoides pteronyssinus preparation results in decreased numbers of IL-4+ T cells and expansion of CD4+IL-10+ T cells expressing a peripheral tissue trafficking phenotype. The co-localization of IL-10+ staining to CD4+CD25+ T cells is consistent with the induction of a T regulatory cell population by SIT.     

Comparison of allergen-stimulated dendritic cells from atopic and nonatopic donors dissecting their effect on autologous naive and memory T helper cells of such donors

BACKGROUND: Because of their production of IL-12, mature dendritic cells (DC) are potent inducers of T(H)1 responses. However, recent reports have demonstrated that DCs can also induce T(H)2 differentiation. OBJECTIVE: In the current study we investigated which immune response is induced by DCs in naive CD45RA(+) or memory CD45R0(+) CD4(+) T cells from atopic individuals (patients with grass pollen, birch pollen, or house dust mite allergy) compared with nonatopic control subjects. METHODS: Immature DCs, generated from peripheral blood monocytes from atopic and nonatopic donors, were pulsed with the respective allergen and fully matured. Then the mature DCs were cocultured in vitro with autologous naive (CD45RA(+)) and memory (CD45R0(+)) CD4(+) T cells and cytokine and IgE production were measured by ELISA. RESULTS: After the second restimulation with allergen-pulsed DCs, naive as well as memory autologous CD4(+) T cells from atopic but not from nonatopic donors showed an enhanced production of the T(H)2-type cytokines IL-4, IL-5, and IL-10, resulting in an increased IgE production, whereas IFN-gamma production and proliferation were not different. IL-12 production and surface marker expression of DCs derived from atopic and nonatopic donors did not differ and addition of neutralizing anti-IL-12 mAbs did not increase IL-4 but diminished IFN-gamma production. CONCLUSION: These data indicate that mature DCs are able to induce naive and activate allergen-specific T helper cells to produce T(H)2 cytokines if the T cells are derived from atopic donors. This phenomenon is not due to diminished IL-12 production by DCs of atopic donors.     

Chemokine responsiveness of CD4+ CD25+ regulatory and CD4+ CD25− T cells from atopic and nonatopic donors

Background:  Allergic inflammation is associated with Th2-type T cells, which can be suppressed by CD4+ CD25+ regulatory T cells (Tregs). Both express chemokine receptors (CCR) 4 and CCR8, but the dynamics of expression and effect of atopic status are unknown.
Objective:  To examine the expression of chemokine receptors by CD4+ CD25+ and CD4+ CD25− T cells from atopic and nonatopic donors, and document response to allergen stimulation in vitro .
Methods:  Chemokine receptor expression was examined by flow cytometry and quantitative PCR of CD4+ CD25hi and CD4+ CD25− T cells from atopics and nonatopics. Responsiveness to chemokines was by actin polymerization. Dynamics of chemokine receptor expression in 6-day allergen-stimulated cultures was analysed by carboxyfluoroscein succinimidyl ester labelling.
Results:  CD4+ CD25hi Tregs preferentially expressed CCR3, CCR4, CCR5, CCR6 and CCR8. CD4+ CD25hi Tregs responded to the chemokine ligands for CCR4, CCR6 and CCR8 (CCL17, 22, 20 and 1 respectively), with no differences between atopic and nonatopic donors. Over 6-day allergen stimulation, CD4+ CD25+ T-cells downregulated CCR4 and upregulated CCR7, in contrast to CD4+ CD25− effector cells, which downregulated CCR7 and upregulated CCR4.
Conclusions:  CCR4, CCR6 and CCR8 have potential roles in localization of both CD4+ CD25+ regulatory and CD4+ CD25− effector T cells to sites of allergic inflammation. Upregulation of CCR7 and downregulation of CCR4 upon allergen stimulation of Tregs may allow their recirculation from sites of inflammation, in contrast to retention of effector T cells.     

Regulatory activity of human CD4 CD25 T cells depends on allergen concentration, type of allergen and atopy status of the donor

Regulatory CD4+ CD25+ FoxP3-positive T cells (Treg) are functional in most atopic patients with allergic rhinitis and are able to inhibit T helper type 1 (Th1) and Th2 cytokine production of CD4+ CD25- T cells. This study was designed to analyse the following additional aspects: influence of allergen concentration, influence of the type of allergen, and influence of the atopy status of the donor on the strength of the regulatory activity. CD4+ CD25- T cells from healthy non-atopic controls or from grass-pollen-allergic or wasp-venom-allergic donors were stimulated alone or in the presence of Treg with autologous mature monocyte-derived dendritic cells which were pulsed with different concentrations of the respective allergens. Treg from grass-pollen-allergic donors failed to inhibit proliferation but not cytokine production of CD4+ CD25- T cells at high antigen doses while Treg from non-atopic donors did not fail at these allergen concentrations. Proliferative responses and cytokine production of CD4+ CD25- T cells from most of the examined wasp-venom-allergic patients were not inhibited at any concentration of wasp venom. The use of wasp venom- or phospholipase A2-pulsed dendritic cells for stimulation of CD4+ CD25- T cells from donors who were not allergic to wasp stings only resulted in an inhibited proliferation and Th2 cytokine production by Treg at 10-fold lower than the optimal concentration, while interferon-gamma production was inhibited at all concentrations investigated. These data demonstrate that in allergic diseases the function of Treg is dependent on the concentration and the type of the respective allergen with different thresholds for individual allergens and patients.     

CD4+CD25+T细胞在系统性红斑狼疮中的表达及意义

目的：探讨CD4^＋ CD25^＋ T细胞、IL-10在系统性红斑狼疮（SLE）患者外周血的表达及临床意义。方法：入组30例SLE患者和20例正常对照者，其中活动性SLE患者17人，非活动性SLE患者13人。用流式细胞仪检测SLE患者和正常对照者的外周血CD4^＋ CD25^＋ T细胞阳性率，用酶联免疫吸附试验（ELISA）检测血清中IL-10浓度。结果：活动性和非活动性SLE患者CD4^＋ T细胞总数均低于正常对照者；活动性和非活动性SLE患者CD4^＋ CD25^＋ T细胞阳性率高于正常对照者；活动性SLE患者IL-10浓度显著高于非活动性SLE患者和正常对照者。SLE患者CD4^＋ CD25^＋ T细胞阳性率和血清IL-10浓度与补体C3、抗DNA抗体水平及SLEDAI积分均无相关性。结论：SLE患者外周血CD4^＋ CD25^＋ T细胞是活化T细胞的标志，IL-10分泌异常与SLE的发病有关。     

Inhibition of allergen-IgE binding to B cells by IgG antibodies after grass pollen immunotherapy

BACKGROUND: Among atopic individuals, levels of allergen-specific IgG antibodies have been inversely associated with the degree of allergen sensitization. Additionally, allergen-specific IgG antibodies are markedly increased by allergen injection immunotherapy. These observations have led to proposals that allergen-specific IgG antibodies might have protective properties in atopic individuals. OBJECTIVE: We hypothesized that after grass pollen immunotherapy, these antibodies disrupt IgE-dependent allergen processing by antigen-presenting cells. METHODS: We have developed a novel flow cytometric assay based on detection of allergen-IgE binding to the low-affinity IgE receptor on B cells to examine the blocking effects of sera collected from 18 patients who participated in a double-blind, controlled trial of grass pollen immunotherapy for 1 year. RESULTS: In all 10 patients who received active therapy, there was induction of activity that inhibited allergen-IgE binding to B cells (P =.02, vs placebo subjects), as well as subsequent allergen presentation to T cells. This activity copurified with IgG and was allergen specific, because sera taken from patients treated with grass pollen immunotherapy but who were also birch pollen sensitive did not inhibit IgE-birch pollen allergen binding to B cells. CONCLUSION: We conclude that allergen-specific IgG antibodies induced by immunotherapy can disrupt formation of allergen-IgE complexes that bind to antigen-presenting cells and facilitate allergen presentation.     

Cytokine production in peripheral blood cells during and outside the pollen season in birch-allergic patients and non-allergic controls

BACKGROUND: The naturally occurring pollen season permits observation of the kinetic changes in the process of allergic inflammation. We examined cytokine production in peripheral blood (PB) T cells and monocytes obtained from birch-allergic patients both during and outside the pollen season. METHODS: PB from 16 patients and six healthy controls was obtained during the alder pollen season, at the beginning and the peak of the birch pollen season and outside the pollen season. Mononuclear cells (MNC) were stimulated with allergen and polyclonal activators. For flow cytometric analysis, MNC were stained with monoclonal antibodies (MoAbs) against the cell surface markers CD3, CD8, CD14 and the intracellular cytokines IL-4, IL-5, IL-10, IL-12, IL-13, granulocyte macrophage-colony stimulating factor (GM-CSF) and IFN-gamma. RESULTS: In allergic patients, significant increases in clinical symptoms, use of medication, eosinophil numbers and birch-specific IgE were found during the pollen season. In vitro allergen stimulation increased the number of GM-CSF+ monocytes (P<0.01) and this increase was dependent on allergen exposure. The IL-4/IFN-gamma ratio rose (P<0.001) at the peak of birch pollen season and the ratio correlated with symptom scores during the birch season. In the CD4+ cell population, the numbers of GM-CSF+ cells were higher throughout the alder and birch seasons compared with outside the pollen season (P<0.05). No such changes were seen in the healthy controls. CONCLUSIONS: The main finding of our study was the increased percentage of GM-CSF+ monocytes in atopic subjects compared with healthy controls. In allergic patients, natural seasonal pollen exposure resulted in increased numbers of GM-CSF+ cells among both monocytes and CD4+ T cells. We have also shown that a seasonal change in Th2/Th1 cytokine ratio requires an adequate and prolonged allergen stimulation that is seen late in the pollen season.     

Grass pollen immunotherapy: symptomatic improvement correlates with reductions in eosinophils and IL-5 mRNA expression in the nasal mucosa during the pollen season

BACKGROUND: Tissue eosinophilia and infiltration by T(H)2-type T cells are characteristic features of allergic rhinitis both after allergen challenge and during natural allergen exposure. Specific immunotherapy inhibits allergen-induced nasal eosinophilia. OBJECTIVES: We sought to assess, in the context of a randomized trial, the relationships between symptomatic improvement after immunotherapy and eosinophil numbers and IL-5 expression in the nasal mucosa during the pollen season. METHODS: Nasal biopsy specimens were taken from 37 adults with severe summer hay fever at baseline (out of season) and at peak season after 2 years of treatment with a depot grass pollen extract or placebo. Biopsy specimens were processed for immunohistochemistry by using mAbs against eosinophils (EG2), T cells (CD3), and IL-2 receptor-positive cells (CD25), as well as for in situ hybridization by using a sulfur 35-labeled antisense riboprobe directed against IL-5. RESULTS: Immunotherapy significantly reduced symptoms (49%, P =.01) and medication requirements (80%, P =.007) compared with placebo. There was a 400% increase (P =.004) in eosinophils during the pollen season in placebo-treated patients, which was inhibited in the immunotherapy group (20% increase, P =.04 between groups). Seasonal increases were also observed for CD25(+) cells (P =.002), CD3(+) cells (P =.02), and IL-5 mRNA-expressing cells (P =.03) in the placebo group but not in the immunotherapy group. A significant correlation was observed between eosinophils and IL-5 expression (r = 0.5, P <.05). Both eosinophils (r = 0.6, P <.02) and IL-5 (r = 0.6, P <.02) correlated with symptoms after immunotherapy. CONCLUSION: Improvement in symptoms after grass pollen immunotherapy may result, at least in part, from inhibition of IL-5-dependent tissue eosinophilia during the pollen season.     

Regulatory CD4+CD25+ T lymphocytes in peripheral blood from patients with atopic asthma

The aim of this study was to analyze whether regulatory CD4+CD25+ T lymphocytes exist and function normally in patients with atopic asthma. Our data showed that a significant increase in CD4+CD25+ cell numbers was seen in atopic asthmatics during acute exacerbation, but not in those stable asthmatics, atopic nonasthmatics, and normal subjects. The mean inhibition values of the proliferation response of CD4+CD25- cells by CD4+CD25+ cells from normal controls and asthmatics were almost the same. There was no difference in inhibitory effects on both Th1 and Th2 cytokine production of CD4+CD25- cells by CD4+CD25+ cells in the two groups. These data demonstrated that although CD4+CD25+ cells increase in atopic asthma during exacerbation, these regulatory T cells appear to function normally with regard to suppression of T-cell proliferation as well as Th1-Th2 cytokine production.