ABSTRACT
Lymphangiogenesis is an important physiological response to inflammatory insult, acting to limit inflammation. Macrophages, dendritic cells, and lymphocytes are known to drive lymphangiogenesis. In this study, we show that neutrophils recruited to sites of inflammation can also coordinate lymphangiogenesis. In the absence of B cells, intranodal lymphangiogenesis induced during prolonged inflammation as a consequence of immunization is dependent on the accumulation of neutrophils. When neutrophils are depleted in wild-type mice developing skin inflammation in response to immunization or contact hypersensitization, lymphangiogenesis is decreased and local inflammation is increased. We demonstrate that neutrophils contribute to lymphangiogenesis primarily by modulating vascular endothelial growth factor (VEGF)-A bioavailability and bioactivity and, to a lesser extent, secreting VEGF-D. We further show that neutrophils increased VEGF-A bioavailability and bioactivity via the secretion of matrix metalloproteinases 9 and heparanase. Together, these findings uncover a novel function for neutrophils as organizers of lymphangiogenesis during inflammation.
Subject(s)
Inflammation/etiology , Inflammation/metabolism , Lymphangiogenesis/physiology , Neutrophils/physiology , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor D/metabolism , Animals , B-Lymphocytes/immunology , Dermatitis/etiology , Dermatitis/metabolism , Dermatitis/pathology , Female , Glucuronidase/metabolism , Inflammation/pathology , Lymphangiogenesis/immunology , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neutrophils/immunology , Neutrophils/pathology , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
BACKGROUND: We previously reported that prior nasal administration of highly attenuated Bordetella pertussis BPZE1 provides effective and sustained protection against lethal challenge with influenza A viruses. The protective effect was mediated by suppressing the production of major pro-inflammatory mediators. To further explore the anti-inflammatory properties of BPZE1, we investigated the effect of BPZE1 nasal pretreatment on two mouse models of allergic disease, allergic airway inflammation, and contact hypersensitivity (CHS). METHODS: Allergic reactions were induced in mice nasally pretreated with live attenuated BPZE1 bacteria using the ovalbumin (OVA)-induced allergic airway inflammation and dinitrochlorobenzene (DNCB)-induced CHS models. RESULTS: Prior BPZE1 nasal treatment suppressed OVA-induced lung inflammation and inflammatory cell recruitment and significantly reduced IgE levels and cytokine production. Similarly, BPZE1 nasal pretreatment markedly inhibited ear swelling, skin inflammation, and production of pro-inflammatory cytokines in the DNCB-induced CHS model. For both models, we showed that BPZE1 pretreatment does not affect the sensitization phase. Upon challenge, BPZE1 pretreatment selectively reduced the level of cytokines whose production is increased and did not affect the basal level of other cytokines. Together, our observations suggest that BPZE1 pretreatment specifically targets those cytokine-producing effector cells that are recruited and involved in the inflammatory reaction. CONCLUSION: Our study demonstrates the broad anti-inflammatory properties of the attenuated B. pertussis BPZE1 vaccine candidate and supports its development as a promising agent to prevent and/or treat allergic diseases.
Subject(s)
Bordetella pertussis/immunology , Dermatitis, Contact/prevention & control , Disease Models, Animal , Pertussis Vaccine/immunology , Pneumonia/prevention & control , Vaccines, Attenuated/immunology , Administration, Intranasal , Animals , Cytokines/metabolism , Dermatitis, Contact/immunology , Dinitrochlorobenzene/immunology , Female , Humans , Mice , Mice, Inbred BALB C , Ovalbumin/immunology , Pertussis Vaccine/administration & dosage , Pneumonia/immunology , Vaccines, Attenuated/administration & dosage , Whooping Cough/immunology , Whooping Cough/prevention & controlABSTRACT
T follicular helper (Tfh) cells were discovered just over a decade ago as germinal centre T cells that help B cells make antibodies. Included in this role is affinity maturation and isotype switching. It is here that their functions become less clear. Tfh cells principally produce IL-21 which inhibits class switching to IgE. Recent studies have questioned whether the germinal centre is the main site of IgE class switching or IgE affinity maturation. In this review, I will examine the evidence that these cells are responsible for regulating IgE class switching and the relationship between Tfh cells and T helper 2 (Th2) effector cells.
Subject(s)
Hypersensitivity/immunology , T-Lymphocytes, Helper-Inducer/immunology , Animals , B-Lymphocytes/immunology , Cell Differentiation , Germinal Center/immunology , Germinal Center/pathology , Humans , Hypersensitivity/metabolism , Immunoglobulin E/immunology , Immunoglobulin E/metabolism , Interleukins/immunology , Interleukins/metabolism , T-Lymphocytes, Helper-Inducer/metabolismABSTRACT
Bronchial mucosal CD8(+) cells are implicated in chronic obstructive pulmonary disease (COPD) pathogenesis, but there are few data on their functional properties. We have developed a novel technique to outgrow these cells from COPD patients in sufficient numbers to examine effector functions. Endobronchial biopsies from 15 COPD smokers and 12 ex-smokers, 11 control smokers and 10 non-smokers were cultured with anti-CD3/interleukin (IL)-2 ± IL-15. Outgrown CD3(+) T cells were characterized in terms of phenotype (expression of CD4, 8, 25, 28, 69 and 56), cytotoxicity and expression of COPD-related cytokines. Compared with IL-2 alone, additional IL-15 increased the yield and viability of biopsy-derived CD3(+) T cells (12-16-day culture without restimulation) without alteration of CD4(+) /CD8(+) ratios or expression of accessory/activation molecules. Biopsy-derived T cells, principally CD8(+)/CD56(+) cells, exhibited statistically significantly greater cytotoxic activity in current or ex-smokers with COPD compared with controls (P < 0·01). Elevated percentages of CD8(+) T cells expressed interferon (IFN)-γ, tumour necrosis factor (TNF)-α and IL-13 (P < 0·01) in current COPD smokers compared with all comparison groups. It is possible to perform functional studies on bronchial mucosal T cells in COPD. We demonstrate increased CD8(+)CD56(+) T cell cytotoxic activity and expression of remodelling cytokines in smokers who develop COPD.
Subject(s)
Cytokines/immunology , Cytotoxicity, Immunologic/immunology , Pulmonary Disease, Chronic Obstructive/immunology , T-Lymphocytes/immunology , Adult , Aged , Biopsy , Bronchi/immunology , Bronchi/pathology , CD3 Complex/immunology , CD3 Complex/metabolism , Cell Culture Techniques/methods , Cell Proliferation/drug effects , Cells, Cultured , Cytokines/metabolism , Female , Flow Cytometry , Humans , Immunophenotyping , Interleukin-12/pharmacology , Interleukin-15/pharmacology , K562 Cells , Male , Middle Aged , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/pathology , Respiratory Mucosa/immunology , Respiratory Mucosa/pathology , Smoking , T-Lymphocytes/drug effects , T-Lymphocytes/metabolismABSTRACT
BACKGROUND: Inhaled endotoxin or lipopolysaccharide (LPS) is implicated in the pathogenesis of pulmonary diseases. We investigated the inhalation effects of two different doses of LPS in healthy human subjects. METHODS: Eighteen healthy non-atopic human subjects inhaled either 15 microg (n=10) or 50 microg (n=8)Escherichia coli LPS in an open study. As control, each subject had isotonic saline inhalation 1 week before (baseline) and after LPS inhalation. Data collected included those of clinical parameter, induced sputum and peripheral blood CD4+ and CD8+ T cells. RESULTS: Acute flu-like symptoms and pyrexia were significantly greater in the 50 microg than 15 microg LPS group. Similarly, the increase in sputum and blood total cell and neutrophil counts at 6h following inhaled LPS were greater in the 50 microg group. Myeloperoxidase, human neutrophil elastase and interleukin-8 in sputum sol, but not blood, showed a trend towards greater increase following 50 microg LPS. All these changes were resolved at one week. In the 50 microg dose group alone, there was a reduction in the proportion of peripheral blood interferon (IFN)-gamma-producing CD4+ and CD8+ T cells at 6h followed by an increase at 1 week after inhaled LPS. CONCLUSIONS: The airway and systemic effects of inhaled LPS are dose-related and predominantly neutrophilic. The changes in the proportions of circulating CD4+ and CD8+ T cells suggests preferential recruitment of IFN-gamma-producing T cells into tissue from inhaled 50 microg LPS, followed by reappearance of these cells in blood 1 week later.
Subject(s)
CD4-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/drug effects , Lipopolysaccharides/adverse effects , Administration, Inhalation , Adult , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Dose-Response Relationship, Drug , Female , Humans , Lipopolysaccharides/administration & dosage , Lymphocyte Count , Male , Sputum/cytologyABSTRACT
In vivo animal models can offer valuable information on several aspects of asthma pathogenesis and treatment. The mouse is increasingly used in these models, mainly because this species allows for the application in vivo of a broad range of immunological tools, including gene deletion technology. Mice, therefore, seem particularly useful to further elucidate factors influencing the response to inhaled allergens. Examples include: the role of immunoregulatory mechanisms that protect against T-helper cell type 2 cell development; the trafficking of T-cells; and the contribution of the innate immunity. However, as for other animal species, murine models also have limitations. Mice do not spontaneously develop asthma and no model mimics the entire asthma phenotype. Instead, mice should be used to model specific traits of the human disease. The present task force report draws attention to specific aspects of lung structure and function that need to be borne in mind when developing such models and interpreting the results. In particular, efforts should be made to develop models that mimic the lung function changes characteristic of asthma as closely as possible. A large section of this report is therefore devoted to an overview of airway function and its measurement in mice.
Subject(s)
Asthma/pathology , Asthma/physiopathology , Disease Models, Animal , Animals , Asthma/immunology , Humans , Lung/immunology , Lung/pathology , Lung/physiopathology , MiceABSTRACT
OBJECTIVES: We have reported that synovial fluid T cells from patients with rheumatoid arthritis (RA) proliferate in response to the endoplasmic reticulum molecular chaperone immunoglobulin binding protein (BiP). The aim of the present work was to clone and define T cells responding to this protein. METHODS: T-cell clones were generated from the peripheral blood of an individual known to respond to BiP by limiting dilution of BiP-stimulated peripheral blood mononuclear cells. T-cell receptor usage of BiP-responsive clones was determined by monoclonal antibody staining followed by flow cytometric analysis. Cytokine production by the BiP-responsive clones was determined by analysis of post-stimulation supernatants by ELISA. Additional phenotyping was performed by flow cytometry. RESULTS: Of 49 clones isolated, six were shown to proliferate in response to BiP. Proliferation was low but consistent. One clone expressed CD4 and five were CD8-positive. Three clones, all CD8(+), grew strongly and were investigated further. T-cell receptor usage was determined in two clones (Vbeta 7.1 and Vbeta 12); the Vbeta element of the remaining clone was not recognized by the panel of antibodies used. All three clones produced interleukin 10 (IL-10) (80-380 pg/ml) and two of them produced IL-4 (10-80 pg/ml) and IL-5 (>5000 pg/ml). One clone produced both IL-10 and interferon gamma (>5000 pg/ml). Additional phenotyping of these clones showed them to express CD25, CD28, CD80 and 86 but not CD56 or 57. One clone constitutively expressed CTLA-4 cytoplasmically. CONCLUSIONS: This study demonstrates that a population of CD8(+) T cells with the cytokine profile of Tc2 cells can be stimulated by the chaperone BiP. These cells may perform a regulatory role in the normal response to inflammation. The increase in response to this antigen in the synovial joint in RA may indicate an attempt to regulate the ongoing inflammation.
Subject(s)
Arthritis, Rheumatoid/immunology , Autoantigens/immunology , CD8-Positive T-Lymphocytes/immunology , Carrier Proteins/immunology , Heat-Shock Proteins , Interleukin-10/biosynthesis , Molecular Chaperones/immunology , Cell Culture Techniques/methods , Cell Division/immunology , Cell Separation/methods , Clone Cells/immunology , Endoplasmic Reticulum Chaperone BiP , Flow Cytometry/methods , Humans , Immunophenotyping , Lymphocyte Activation/immunology , Synovial Fluid/immunology , T-Lymphocyte Subsets/immunologyABSTRACT
Human primary effector T cells were analyzed for their susceptibility to anti-CD3-induced activation-induced cell death (AICD). Th1 and Tc1 cells were more susceptible to AICD than their type 2 counterparts. Type 1 and type 2 subsets were also found to be differentially susceptible to CD95-mediated apoptosis, although cell-surface expression of CD95 and CD95L was at similar levels on all subsets. A role for CD95 in AICD was confirmed by the addition of anti-CD95L antibodies that partially abrogated AICD. Residual apoptosis could not be accounted for by TNF-alpha/TNFR interactions because although type 1 cells secreted more TNF-alpha than type 2 cells, the addition of TNFR:Fc fusion protein did not inhibit AICD. Instead, a reduction in AICD was observed in the presence of EGTA or concanamycin A. The inhibition of apoptosis by a granzyme B inhibitor z-AAD-CMK in Tc1 cells further indicated an involvement of the granule exocytosis mechanism in AICD.
Subject(s)
Lymphocyte Activation , Macrolides , Membrane Glycoproteins/physiology , Serine Endopeptidases/physiology , T-Lymphocyte Subsets/cytology , fas Receptor/physiology , Adult , Amino Acid Chloromethyl Ketones/pharmacology , Anti-Bacterial Agents/pharmacology , Calcium , Caspase 8 , Caspase 9 , Caspase Inhibitors , Chelating Agents/pharmacology , Cytoplasmic Granules/metabolism , Egtazic Acid/pharmacology , Enzyme Inhibitors/pharmacology , Etanercept , Exocytosis , Fas Ligand Protein , Granzymes , Humans , Immunoglobulin G/pharmacology , Interferon-gamma/metabolism , Interleukin-12/pharmacology , Interleukin-2/pharmacology , Interleukin-4/metabolism , Membrane Glycoproteins/antagonists & inhibitors , Muromonab-CD3/pharmacology , Receptors, Tumor Necrosis Factor/physiology , Recombinant Fusion Proteins/physiology , Recombinant Proteins/pharmacology , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Th1 Cells/cytology , Th1 Cells/drug effects , Th1 Cells/immunology , Th1 Cells/metabolism , Th2 Cells/cytology , Th2 Cells/drug effects , Th2 Cells/immunology , Th2 Cells/metabolism , Tumor Necrosis Factor-alpha/physiologyABSTRACT
Differentiation of developing T cells into the type 1 (IFN-gamma-producing) or type 2 (IL-4-producing) subsets is a central theme of immune regulation. The balance of IL-4 and IL-12 present during T cell activation has been considered the major influence on type 1 versus type 2 development. Here we show that CD4 T cells can become biased towards type 1 or type 2 phenotypes during their initial activation in the absence of IL-4 or IL-12. This type of regulation is dependent on the balance of MAPkinase, protein kinase C, and calcineurin signaling after TCR engagement. Later maturation of Th1 or Th2 effectors is dependent on IL-12 or IL-4. However Tc1 CD8 effector development is independent of IL-12, and Tc2 cell generation requires both appropriate TCR signals and IL-4 early in effector development. Using an altered peptide ligand to stimulate TCR transgenic T cells, we show that altered signaling regulates the numbers of CD8 cells capable of developing into Tc2 effectors, and also their responsiveness to IL-4. Together, the results support a two-stage model of differentiation in which intermediate cells biased towards the type 1 or type 2 pathways after activation, are subsequently matured in response to IL-12 or IL-4, respectively.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Interleukin-12/physiology , Interleukin-4/physiology , Receptors, Antigen, T-Cell/immunology , Th1 Cells/immunology , Th2 Cells/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , Cell Differentiation , Cells, Cultured , Cytokines/biosynthesis , Enzyme Inhibitors/pharmacology , Interferon-gamma/biosynthesis , Interleukin-12/pharmacology , Interleukin-4/biosynthesis , Interleukin-4/pharmacology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Peptides/immunology , Phenotype , Signal TransductionABSTRACT
BACKGROUND: It is well recognized that CD8 T cells inhibit IgE responses. In this study, we investigated the mechanism of CD8 T cell-mediated IgE suppression by comparing the capacity of T cytotoxic 1 (Tc1) and T cytotoxic 2 (Tc2) CD8 T cells to inhibit IgE responses to ovalbumin (OVA). METHODS: Tc1 and Tc2 CD8 T cells were generated from OVA(257-264)-specific Vbeta5.2 T cell receptor (TcR) transgenic mice by stimulation with anti-CD3 and anti-CD28 under Tc1 and Tc2 polarizing conditions. Tc1 and Tc2 Vbeta5.2 TcR CD8 T cells (10(6)) were adoptively transferred to syngeneic mice, and following immunization with 100 micro of OVA/alum, serum IgE antibodies were measured by passive cutaneous anaphylaxis and expressed as the highest dilution that gave a detectable skin response. RESULTS: Both Tc1 and Tc2 CD8 T cells from OT-I mice inhibited IgE. CONCLUSION: Both Tc1 and Tc2 CD8 T cells promote Th1 immunity and inhibit IgE responses. This process appears to be independent of CD8 T cell-derived IFN-gamma, as both Tc2 (IFN-gamma-) and Tc1 (IFN-gamma+) CD8 T cells inhibited IgE.
Subject(s)
Immunoglobulin E/biosynthesis , T-Lymphocytes, Cytotoxic/immunology , Adoptive Transfer , Allergens/immunology , Animals , Cells, Cultured , Genes, T-Cell Receptor , Immunoglobulin Variable Region/genetics , Interferon-gamma/biosynthesis , Interleukin-4/biosynthesis , Mice , Mice, Inbred C57BL , Mice, Transgenic , Ovalbumin/immunology , T-Lymphocytes, Cytotoxic/classification , T-Lymphocytes, Cytotoxic/transplantationABSTRACT
Th2 T cell immune-driven inflammation plays an important role in allergic asthma. We studied the effect of counterbalancing Th1 T cells in an asthma model in Brown Norway rats that favors Th2 responses. Rats received i.v. transfers of syngeneic allergen-specific Th1 or Th2 cells, 24 h before aerosol exposure to allergen, and were studied 18-24 h later. Adoptive transfer of OVA-specific Th2 cells, but not Th1 cells, and OVA, but not BSA exposure, induced bronchial hyperresponsiveness (BHR) to acetylcholine and eosinophilia in a cell number-dependent manner. Importantly, cotransfer of OVA-specific Th1 cells dose-dependently reversed BHR and bronchoalveolar lavage (BAL) eosinophilia, but not mucosal eosinophilia. OVA-specific Th1 cells transferred alone induced mucosal eosinophilia, but neither BHR nor BAL eosinophilia. Th1 suppression of BHR and BAL eosinophilia was allergen specific, since cotransfer of BSA-specific Th1 cells with the OVA-specific Th2 cells was not inhibitory when OVA aerosol alone was used, but was suppressive with OVA and BSA challenge. Furthermore, recipients of Th1 cells alone had increased gene expression for IFN-gamma in the lungs, while those receiving Th2 cells alone showed increased IL-4 mRNA. Importantly, induction of these Th2 cytokines was inhibited in recipients of combined Th1 and Th2 cells. Anti-IFN-gamma treatment attenuated the down-regulatory effect of Th1 cells. Allergen-specific Th1 cells down-regulate efferent Th2 cytokine-dependent BHR and BAL eosinophilia in an asthma model via mechanisms that depend on IFN-gamma. Therapy designed to control the efferent phase of established asthma by augmenting down-regulatory Th1 counterbalancing mechanisms should be effective.
Subject(s)
Allergens/immunology , Bronchial Hyperreactivity/prevention & control , Epitopes, T-Lymphocyte/immunology , Interferon-gamma/physiology , Pulmonary Eosinophilia/pathology , Pulmonary Eosinophilia/prevention & control , Th1 Cells/immunology , Th2 Cells/immunology , Administration, Inhalation , Adoptive Transfer , Allergens/administration & dosage , Animals , Antibodies, Monoclonal/administration & dosage , Bronchial Hyperreactivity/immunology , Bronchial Hyperreactivity/pathology , Bronchoalveolar Lavage Fluid/immunology , Cell Line , Epitopes, T-Lymphocyte/administration & dosage , Inflammation/immunology , Inflammation/pathology , Inflammation/prevention & control , Injections, Intravenous , Interferon-gamma/immunology , Interleukin-4/antagonists & inhibitors , Interleukin-4/genetics , Lung/cytology , Lung/immunology , Male , Ovalbumin/administration & dosage , Ovalbumin/immunology , Pulmonary Eosinophilia/immunology , RNA, Messenger/antagonists & inhibitors , Rats , Rats, Inbred BN , Th1 Cells/transplantation , Th2 Cells/transplantationABSTRACT
An increase in the prevalence of allergy and allergic diseases has taken place in the industrialised countries. Allergic diseases represent a major health problem, and appear linked to affluence and modern lifestyle. In the 20th century air pollution from industrial sources largely has been replaced by diesel exhaust and other traffic pollution. Further, the indoor environment in which we spend most of our time has changed dramatically. In order to understand the contribution of pollution and other environmental changes to the development of allergy, we need to understand the biologic processes that underlie allergic immune responses. In the present paper, immune regulatory pathways that control the allergic immune response are delineated. Castor bean dust causes widespread allergic sensitisation. The investigations that made clear the importance of CD8 T cells for the regulation of IgE production were triggered by studies of castor bean allergy. A special focus is in this review placed on the regulatory role of CD8 T cells in the development of the allergic immune response.
Subject(s)
Environmental Pollutants/adverse effects , Hypersensitivity/etiology , Animals , Cell Differentiation , Cytokines/biosynthesis , Humans , Immunoglobulin E/biosynthesis , T-Lymphocyte Subsets/immunologyABSTRACT
BACKGROUND: Topical exposure to chemical allergens such as trimellitic anhydride or 1-chloro-2,4-dinitrochlorobenzene results in the accumulation of dendritic cells (DCs) and subsequent rapid up-regulation of CD4 T-cell proliferation and cytokine secretion within draining lymph nodes. OBJECTIVE: We investigated the contribution of antigen-specific and CD40 ligand (CD40L)-mediated signals to chemical allergen-induced CD4 T-cell growth and cytokine production. METHODS: DCs enriched from lymph nodes of allergen-challenged animals by metrizamide centrifugation were used to stimulate cytokine and proliferative responses by magnetic immunobead-sorted CD4 T cells primed in vivo with the same or unrelated allergen. Cultures of DCs and T cells were supplemented with antibodies that block IL-12 and CD40L activity. RESULTS: Proliferation of CD4 T cells was stimulated by DCs primed with the same but not unrelated antigen, whereas IFN-gamma, IL-12, and IL-10 secretion were provoked equally well by DCs primed with either hapten. Blockade of CD40L engagement abrogated production of IFN-gamma (80%) and IL-12 (95%) under antigen-nonspecific stimulatory conditions. In contrast, IL-10 secretion was enhanced after CD40L blockade under both antigen-specific and nonspecific conditions. Primary CD4 T cells activated by mitogen were also influenced by DCs in the same way. CONCLUSION: These results show that during the development of chemical sensitization emerging CD4 T-cell growth and cytokine production are regulated by independent mechanisms requiring antigen presentation and CD40 signaling, respectively.
Subject(s)
Cytokines/biosynthesis , Dinitrochlorobenzene/immunology , Epitopes/immunology , Phthalic Anhydrides/immunology , Allergens , Animals , Antibodies/therapeutic use , CD4-Positive T-Lymphocytes/immunology , CD40 Antigens/pharmacology , CD40 Ligand , Cytokines/immunology , Dendritic Cells/drug effects , Dendritic Cells/immunology , Drug Interactions , Female , Immunization , Interferon-gamma/biosynthesis , Interleukin-10/biosynthesis , Interleukin-12/biosynthesis , Interleukin-12/immunology , Ligands , Lymphocyte Activation , Membrane Glycoproteins/pharmacology , Mice , Mice, Inbred BALB CABSTRACT
BACKGROUND: The role of Immunoglobulin (Ig)E in inflammation is the subject of considerable study and a number of studies have shown conflicting evidence for its role in eosinophil recruitment and bronchial hyperresponsiveness in a number of murine models. The low affinity IgE receptor, CD23, is known to act as a negative regulator of IgE production and we have used knockout mice deficient in CD23 to investigate the role of IgE in eosinophil recruitment and bronchial hyperresponsiveness in a murine model of airway inflammation. OBJECTIVE: To study the role of the low affinity FcepsilonII receptor, CD23 in IgE production, lung inflammation and bronchial hyperresponsiveness. METHODS: Wild-type and CD23 knockout C57Bl/6 mice (CD23-/-) were immunized by intraperitoneal injection with ovalbumin on days 0 and 14 and challenged with aerosolized antigen on day 21 for a period of up to 1 week. Blood samples, bronchoalveolar lavage and lung tissue samples were obtained to determine serum IgE levels and inflammatory cell numbers, respectively. Furthermore, airway resistance was measured to increasing concentrations of aerosolized 5-hydroxytryptamine in order to evaluate the effect of CD23 deficiency on bronchial hyperresponsiveness to antigen challenge. RESULTS: Sensitization of wild-type C57Bl/6 mice to ovalbumin resulted in elevated levels of total serum IgE and ovalbumin-specific IgE, which was significantly augmented in CD23 knockout C57Bl/6 mice (CD23-/-). A significant increase in the percentage of eosinophils recovered in bronchoalveolar lavage fluid from wild-type and CD23-/- mice was observed 24 h following 3 or 7 days aerosol exposure with ovalbumin (10 mg/mL). At 3 days, the increase in the percentage of eosinophils was significantly greater in CD23-/- groups. Immunohistochemical analysis of lungs sections revealed the presence of CD3+, CD4+ and CD23+ cells in wild-type mice but a lack of immunofluorescence of CD23+ cells in CD23-/- mice. In wild-type ovalbumin-immunized mice, bronchial hyperresponsiveness to aerosolized 5-hydroxytryptamine was observed following a 3-day antigen challenge, which was significantly greater in CD23-/- ovalbumin-immunized mice. CONCLUSION: These studies demonstrate that CD23-/- mice have increased capacity to produce IgE consistent with the view of a negative feedback role for membrane-bound CD23 and under such conditions, may account for the greater numbers of eosinophils recruited to the airways and bronchial hyperresponsiveness observed following acute but not chronic antigen challenge.
Subject(s)
Allergens/administration & dosage , Bronchial Hyperreactivity/immunology , Immunoglobulin E/metabolism , Pulmonary Eosinophilia/immunology , Receptors, IgE/physiology , Animals , Bronchial Hyperreactivity/metabolism , Bronchial Hyperreactivity/pathology , Bronchoalveolar Lavage Fluid/immunology , Immunoglobulin E/blood , Mice , Mice, Inbred C57BL , Mice, Knockout , Pulmonary Eosinophilia/metabolism , Pulmonary Eosinophilia/pathology , Respiratory Mucosa/immunology , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathologyABSTRACT
Viruses cause asthmatic exacerbations in schoolchildren. We tested the hypothesis that children who wheezed with viral respiratory tract infections secrete higher levels of the type 1 cytokine interferon-gamma (IFN-gamma) in the peripheral circulation than children who had never wheezed. Blood was taken from 13 children (eight atopic) with episodic wheeze and 11 controls. CD4 and CD8 cells were separated from peripheral blood mononuclear cells and stimulated with phorbol 12-myrisate 13-acetate (PMA) and ionomycin for 24 h. IFN-gamma, IL-4, and IL-5 were measured in the supernatant by ELISA. IFN-gamma production by CD4 and CD8 cells was lower in children with a history of wheeze (CD4, P = 0.046; CD8, P = 0.037). These children were then analysed according to atopic status. CD4 and CD8 IFN-gamma production in nonatopic wheezy children was reduced (CD4, P=0.009; CD8, P=0.003). IFN-gamma production by atopic wheezy children was lower than by controls, but the differences were not significant (CD4, P = 0.2831; CD8, P = 0.1372). CD8 IL-5 was lower in children who wheezed (P=0.012). Release of IL-4 and IL-5 by CD4 cells did not differ between the three groups. We propose that defective IFN-gamma secretion by CD4 and CD8 cells may contribute to viral-induced wheeze in nonatopic children.
Subject(s)
Interferon-gamma/metabolism , Respiratory Sounds/etiology , Respiratory Tract Infections/complications , Virus Diseases/complications , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Cells, Cultured , Child , Enzyme-Linked Immunosorbent Assay , Humans , Hypersensitivity, Immediate/immunology , Interleukin-4/metabolism , Interleukin-5/metabolismABSTRACT
Development of naive T cells into type 1 (Th1, Tc1) or type 2 (Th2, Tc2) effector cells is thought to be under the control of cytokines. In this study, we show that when both IL-12 and IL-4 are present, murine and human T cell differentiation is regulated by the balance of protein kinase C (PKC) and calcium signaling within T cells. Although both biochemical signals were required for T cell activation via the TCR, altering the balance between them redirected type 1 cells to type 2 and vice versa. Stimulation of calcium signaling or inhibition of PKC favored type 1 differentiation, whereas stimulation of PKC or inhibition of calcineurin resulted in type 2 effectors. Altered peptide ligands induced distinct balances of PKC/calcium signaling and altered Tc1/Tc2 development in TCR-transgenic CD8 T cells. The data suggest novel strategies for manipulation of the immune response in vivo.
Subject(s)
Calcium Signaling/immunology , Protein Kinase C/physiology , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/enzymology , Animals , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/enzymology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/enzymology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Calcineurin/physiology , Calcium Signaling/drug effects , Cell Differentiation/drug effects , Cell Differentiation/immunology , Clone Cells , Humans , Ionomycin/pharmacology , Ligands , Lymphocyte Activation/drug effects , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Receptors, Antigen, T-Cell/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/enzymology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Th2 Cells/drug effects , Th2 Cells/enzymology , Th2 Cells/immunology , Th2 Cells/metabolismABSTRACT
A characteristic feature of allergic asthma is the overexpression of the T helper type 2 (Th2) cytokines interleukin-4 (IL-4), IL-5 and IL-13 by T lymphocytes. Of these cytokines, IL-5 is critical for the growth, survival and recruitment of eosinophils which are thought to be responsible for the tissue damage observed in asthmatic airways. The expression of human IL-5 is primarily regulated at the transcriptional level; however, little is known about the mechanisms that control its transcription. Using nuclear extracts from allergen-specific human T-cell clones we have performed DNase I footprinting of the human IL-5 promoter in order to establish sites occupied by transcription factors. We show footprints covering the conserved lymphokine element 0 ¿(CLE0) -60 to -44 base pairs (bp) and GATA (-73 to -62 bp) elements, which have previously been identified to be important in the regulation of the murine IL-5 promoter. We also describe a footprint covering a considerably extended Octamer binding site (-249 to -217 bp), which encompasses two hitherto unidentified CCAAT/enhancer binding protein consensus binding sites. We have also identified a previously unknown Ets binding site (-274 to -264 bp). These novel data on the regions of the human IL-5 promoter that are bound by transcription factors should allow dissection of the regulatory mechanisms involved in the transcription of IL-5 in the T-helper lymphocytes of asthmatics.
Subject(s)
Asthma/immunology , Gene Expression Regulation , Interleukin-5/genetics , Promoter Regions, Genetic , T-Lymphocytes/immunology , Binding Sites , Clone Cells , DNA Footprinting , Deoxyribonuclease I , Humans , Reverse Transcriptase Polymerase Chain Reaction , Transcription FactorsABSTRACT
It has recently become clear that distinct subsets of CD8 T cells, analogous to their CD4 counterparts, exist in rodents and humans. To examine functional differences between human CD8 T-cell subsets, we generated Tc1, Tc2, and Tc0 T-cell clones from the peripheral blood of healthy individuals. The majority of CD8 T-cell clones generated displayed a classic Tc1 phenotype, but 10% to 20% secreted interleukin (IL)-4 in addition to interferon-gamma (Tc0 phenotype). Generation of Tc2 clones was dependent on the use of anti-CD3 and anti-CD28 as the primary stimulus. The cytokine profiles of established clones remained susceptible to modification by the addition of IL-12 and IL-4. In addition, IL-12 enhanced and IL-4 inhibited the growth of Tc1 but not Tc2/0 CD8 T-cell clones. Significant functional differences were observed between the subsets. Tc2/0 clones expressed CD30 and CD40 ligand at a much higher level than Tc1 clones. Both Tc1 and Tc2/0 clones showed comparable cytotoxicity and produced similar levels of perforin and Fas L. However, Tc2 clones were much more resistant to activation-induced cell death and less susceptible to apoptosis by direct Fas ligation. Moreover, Tc1 and Tc2 clones had opposing effects on the development of CD4 effectors, promoting type 1 and type 2 responses, respectively. These data provide evidence for profound differences between human CD8 T-cell subsets that may be important in their functions as cytotoxic or immunoregulatory cells. (Blood. 2000;95:231-240)
Subject(s)
Antigens, CD/biosynthesis , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Cytokines/biosynthesis , Antigens, CD/analysis , Apoptosis/drug effects , CD4-CD8 Ratio , CD4-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/drug effects , Cell Differentiation , Clone Cells , Coculture Techniques , Cytotoxicity, Immunologic , Fas Ligand Protein , Flow Cytometry , Humans , Immunophenotyping , Interleukin-12/pharmacology , Interleukin-4/pharmacology , Ionomycin/pharmacology , Lymphocyte Activation , Membrane Glycoproteins/biosynthesis , Perforin , Pore Forming Cytotoxic Proteins , Reference Values , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
Interferon-gamma (IFN-gamma) is an important cytokine involved in the regulation of allergen-induced immune responses. We examined the role of IFN-gamma in a Brown-Norway rat model of bronchial hyperresponsiveness (BHR) and airway eosinophilia, and its effects on the mRNA expression of T helper type 1 (Th1)/Th2 cytokine. Ovalbumin (OA)-sensitized animals were given either exogenous IFN-gamma (105 U/rat over 3 days, intraperitoneally) or anti-IFN-gamma blocking antibody (DB-1 0.3 mg/rat, intravenously) prior to exposure to OA aerosol and were studied 18-24 hr later. In sensitized animals, OA induced significant BHR, accumulation of eosinophils, T lymphocytes and neutrophils in bronchoalveolar lavage (BAL) fluid, and also increased eosinophils and CD8+ T cells in the airways. Exogenous IFN-gamma attenuated allergen-induced BHR (P<0.02, compared with sham-treated animals) together with a significant reduction in eosinophil and neutrophil numbers in BAL fluid (P<0. 005), and eosinophils and CD8+ T cells in airways (P<0.05). By contrast, anti-IFN-gamma antibody increased airway CD4+ T cells and BHR. Using reverse transcriptase-polymerase chain reaction, significant increases in Th2 [interleukin-4 (IL-4), IL-5 and IL-10], and IFN-gamma cytokine mRNA were found in the lungs of sensitized and OA-exposed animals, while exogenous IFN-gamma significantly suppressed IL-4, IL-5 and IL-10 mRNA expression, and anti-IFN-gamma antibody increased IL-4 and IL-5 mRNA expression. These results indicate that Th1 effects, such as those mediated by IFN-gamma, play a down-regulatory role to suppress the Th2 responses associated with allergen-induced BHR and eosinophilic inflammation.