Eosinophil Growth Research Articles

Dasatinib Inhibits the Growth of Neoplastic Human Eosinophils (EOL-1) through Targeting of FIP1L1-PDGFRα.

Chronic eosinophilic leukemia (CEL) is a myeloproliferative disorder characterized by molecular and/or cytogenetic evidence of monoclonality of eosinophils, sustained marked eosinophilia, and consecutive organ damage. In a majority of patients with CEL with or without associated mastocytosis, the transforming mutation FIP1L1-PDGFRα and the related CHIC2 deletion is found. The respective oncoprotein, FIP1L1-PDGFRα, is considered to play a major role in malignant cell growth in CEL. The tyrosine kinase (TK) inhibitor imatinib (STI571) has been described to counteract the TK activity of FIP1L1-PDGFRα in most patients, and has been introduced as a novel effective therapy in CEL. However, not all patients with CEL show a response to imatinib. Therefore, several attempts have been made to identify other TK inhibitors that counteract growth of neoplastic eosinophils in CEL. We provide evidence that dasatinib, a multi-targeted kinase inhibitor, blocks the growth and survival of EOL-1, an eosinophil leukemia cell line carrying FIP1L1-PDGFRα. The effects of dasatinib on proliferation of EOL-1 cells were dose-dependent, with an IC50 of 0.5–1 nM, that was found to be in the same range compared to IC50 values produced by imatinib. Dasatinib was also found to induce apoptosis in EOL-1 cells in a dose-dependent manner (IC-50: 1–10 nM). The apoptosis-inducing effects of dasatinib on EOL-1 cells were demonstrable by light microscopy, flow cytometry, and by a Tunel assay. To further examine the mechanism of growth inhibition induced by dasatinib in neoplastic eosinophils, Western blot experiments were performed using antibodies directed against phosphorylated or total PDGFRα. In these experiments, we were able to show that dasatinib at 1 μM completely blocks the phosphorylation of FIP1L1-PDGFRα in EOL-1 cells. In summary, our data show that dasatinib inhibits the growth of leukemic eosinophils through targeting of the TK activity of the disease-related oncoprotein FIP1L1-PDGFRα. Based on this observation, dasatinib may be considered as a new interesting treatment option for patients with CEL. As dasatinib is also known to block various KIT mutants as well as wild type KIT, such therapy may also be of interest for patients who have systemic mastocytosis (SM) with an associated CEL (SM-CEL).

Eosinophils from Lineage-Ablated ΔdblGATA Bone Marrow Progenitors: The dblGATA Enhancer in the Promoter of GATA-1 Is Not Essential for Differentiation Ex Vivo

A critical role for eosinophils in remodeling of allergic airways was observed in vivo upon disruption of the dblGATA enhancer that regulates expression of GATA-1, which resulted in an eosinophil-deficient phenotype in the DeltadblGATA mouse. We demonstrate here that bone marrow progenitors isolated from DeltadblGATA mice can differentiate into mature eosinophils when subjected to cytokine stimulation ex vivo. Cultured DeltadblGATA eosinophils contain cytoplasmic granules with immunoreactive major basic protein and they express surface Siglec F and transcripts encoding major basic protein, eosinophil peroxidase, and GATA-1, -2, and -3 to an extent indistinguishable from cultured wild-type eosinophils. Fibroblast coculture and bone marrow cross-transplant experiments indicate that the in vivo eosinophil deficit is an intrinsic progenitor defect, and remains unaffected by interactions with stromal cells. Interestingly, and in contrast to those from the wild type, a majority of the GATA-1 transcripts from cultured DeltadblGATA progenitors express a variant GATA-1 transcript that includes a first exon (1E(B)), located approximately 3700 bp downstream to the previously described first exon found in hemopoietic cells (1E(A)) and approximately 42 bp upstream to another variant first exon, 1E(C). These data suggest that cultured progenitors are able to circumvent the effects of the DeltadblGATA ablation by using a second, more proximal, promoter and use this mechanism to generate quantities of GATA-1 that will support eosinophil growth and differentiation.

Concordant Modulation of Cysteinyl Leukotriene Receptor Expression by IL-4 and IFN-γ on Peripheral Immune Cells

Arachidonic acid can be metabolized to form a group of compounds known as the cysteinyl leukotrienes (CysLT) that bind to one of two receptors to mediate their actions. On circulating cells, expression of the leukotriene receptors is low, but in inflamed tissue the receptor number is dramatically increased. We hypothesized that the cytokine milieu present during inflammation can increase receptor expression on infiltrating immune cells. Various cell populations were purified from peripheral blood and stimulated in vitro with cytokines characteristic of allergic inflammatory disorders, and CysLT receptor expression was measured using quantitative PCR analysis, Western blot, and flow cytometry. IL-4, but not IL-13, was able to significantly induce mRNA and protein levels for both CysLT receptor 1 and 2 from T cells and B cells. CysLT2 receptor expression was also significantly increased in monocytes and eosinophils after IL-4 stimulation. Surprisingly, CysLT2 receptor expression was increased in monocytes, T cells, and B cells when IFN-gamma was used as the stimulus. Factors involved in eosinophil growth and survival were tested for their ability to alter CysLT receptor expression. These results support the concept that cytokines increase expression of both receptors on lymphocytes and granulocytes, allowing these cells to be more responsive to secreted leukotrienes at sites of inflammation.

Humanized Anti-IL-5 Therapy (Mepolizumab) Reduces Eosinophil Responsiveness to the Eotaxins

RATIONALE: IL-5 positively regulates eosinophil growth, activation, survival and tissue recruitment. As such, humanized monoclonal anti-IL-5 antibody (mepolizumab) may decrease activation of eosinophils in response to the eotaxins.METHODS: Seven hypereosinophilic patients received anti-IL-5 therapy (three monthly doses of 750 mg i.v.). Blood and tissue eosinophil levels were determined. Blood eosinophil shape changes in response to the eotaxins were analyzed (via FACS) before and after anti-IL-5.RESULTS: Anti-IL-5 therapy decreased peripheral blood eosinophilia (including the number of CCR3+ cells) by 6-10-fold. Significant decreases in eosinophil shape change responses to eotaxin-1 at concentrations of 10, 30 and 100 ng/ml and to eotaxin-2 at concentrations of 30 and 100 ng/ml were observed (from 38 ± 10, 40 ± 7 and 34 ± 10 (mean relative percentage ± SD) to 24 ± 7, 24 ± 5 and 22 ± 3, and from 41 ± 10 and 43 ± 9 to 20 ± 3 and 27 ± 9, for eotaxin-1 and eotaxin-2, respectively; p<0.05). A similar trend was observed for eotaxin-3 at concentrations of 30 and 100 ng/ml (from 28 ± 12 and 33 ± 10 to 19 ± 2 and 24 ± 9, before and after treatment, respectively).CONCLUSIONS: As such, in addition to lowering blood eosinophilia, humanized anti-IL-5 therapy significantly reduces eosinophil activation responses to the eotaxins. RATIONALE: IL-5 positively regulates eosinophil growth, activation, survival and tissue recruitment. As such, humanized monoclonal anti-IL-5 antibody (mepolizumab) may decrease activation of eosinophils in response to the eotaxins. METHODS: Seven hypereosinophilic patients received anti-IL-5 therapy (three monthly doses of 750 mg i.v.). Blood and tissue eosinophil levels were determined. Blood eosinophil shape changes in response to the eotaxins were analyzed (via FACS) before and after anti-IL-5. RESULTS: Anti-IL-5 therapy decreased peripheral blood eosinophilia (including the number of CCR3+ cells) by 6-10-fold. Significant decreases in eosinophil shape change responses to eotaxin-1 at concentrations of 10, 30 and 100 ng/ml and to eotaxin-2 at concentrations of 30 and 100 ng/ml were observed (from 38 ± 10, 40 ± 7 and 34 ± 10 (mean relative percentage ± SD) to 24 ± 7, 24 ± 5 and 22 ± 3, and from 41 ± 10 and 43 ± 9 to 20 ± 3 and 27 ± 9, for eotaxin-1 and eotaxin-2, respectively; p<0.05). A similar trend was observed for eotaxin-3 at concentrations of 30 and 100 ng/ml (from 28 ± 12 and 33 ± 10 to 19 ± 2 and 24 ± 9, before and after treatment, respectively). CONCLUSIONS: As such, in addition to lowering blood eosinophilia, humanized anti-IL-5 therapy significantly reduces eosinophil activation responses to the eotaxins.

Anti–IL-5 (mepolizumab) therapy for eosinophilic esophagitis

Eosinophilic esophagitis (EE) is characterized by high numbers of eosinophils in the esophagus and epithelial hyperplasia, and is being increasingly recognized. IL-5 promotes eosinophil trafficking to the esophagus, and positively regulates eosinophil growth, activation, survival, and tissue recruitment. We hypothesized that the humanized monoclonal IgG(1) antibody against human IL-5 (mepolizumab) may be useful in the control of EE. An open-label phase I/II safety and efficacy study of anti-IL-5 in 4 adult patients with EE and longstanding dysphagia and esophageal strictures was conducted. Patients received 3 infusions of anti-IL-5 (750 mg intravenously monthly) without change in their current therapy. The levels of plasma IL-5, peripheral blood eosinophils, and CCR3+ cells in blood, quality of life measurements, and histological analysis of esophageal biopsies were determined before and 1 month after treatment. Peripheral blood eosinophilia and percent of CCR3+ cells decreased by 6.4-fold and 7.9-fold (P < .05), respectively, after anti-IL-5 treatment. Notably, mean and maximal esophageal eosinophilia decreased from 46 to 6 and from 153 to 28 eosinophils/high-power field (x400; average, 8.9-fold, P < .001, and 6-fold, P < .05), respectively. Patients reported a better clinical outcome and improved quality of life (P = .03). Therapy was generally well tolerated, and responsiveness to anti-IL-5 therapy did not correlate with plasma IL-5 levels. Anti-IL-5 therapy is associated with marked decreases in peripheral blood and esophageal eosinophilia (including the number of CCR3+ blood cells) in patients with EE and improved clinical outcomes. Anti-IL-5 is a promising therapeutic intervention for EE.

Potential of Blood Eosinophils, Eosinophil-Derived Neurotoxin, and Eotaxin-3 as Biomarkers of Eosinophilic Esophagitis

Eosinophilic esophagitis (EE) is an increasingly recognized disorder characterized by eosinophilic inflammation of the esophageal mucosa, and typically requires serial invasive endoscopic biopsy examinations to document the characteristic histologic features of the disorder. The aim of this study was to identify noninvasive biomarkers that correlated with disease activity and response to treatment as measured by esophageal eosinophilia. A prospective, cross-sectional analysis was performed on 47 pediatric patients undergoing endoscopic evaluation of possible EE. Blood samples were collected for measurement of peripheral blood absolute eosinophil count (AEC) and levels of eosinophil-derived neurotoxin (EDN), eotaxin-1, -2, and -3, and interleukin-5. Stool samples were collected for measurement of EDN. Biomarker levels were correlated with esophageal eosinophil density, and differences in biomarker levels based on disease activity and treatment were determined. AEC, plasma EDN levels, and eotaxin-3 levels significantly correlated with esophageal eosinophil density (AEC: r = 0.56, P < .0001; EDN: r = 0.54, P < .0001; eotaxin-3: r = 0.32, P = .04), and were increased in patients with active EE vs controls (AEC: 440 vs 140 eosinophils/muL, P < .05; EDN: 50.3 vs 31.1 ng/mL, P = .01; eotaxin-3: 37.7 vs 11.5 pg/mL, P = .01). Cut-off values were established to maximize the sensitivity, specificity, and predictive values of these biomarkers alone and in combination. Eotaxin-1, eotaxin-2, interleukin-5, and fecal EDN levels did not correlate with esophageal eosinophil density, and were not increased in active EE vs controls or those with inactive EE. These data show that blood levels of AEC, EDN, and eotaxin-3 may have value as noninvasive biomarkers for monitoring EE.

Immunopathology of atopic dermatitis

Immunopathology of atopic dermatitis

Hypereosinophilic syndromes and new therapeutic approaches including anti-IL-5

Eosinophilia syndromes are heterogenous disorders possessing different clinical manifestations characterized by peripheral blood eosinophilia and end organ damage. Treatment includes glucocorticoids, hydroxyurea (hydroxycarbamide), chemotherapy and interferon-α. Recently, an explanation for the eosinophilia has been found in many patients who would previously have been regarded as having an idiopathic hypereosinophilic syndrome. Such cases have fallen into myeloproliferative and lymphoid categories with some cases remaining unexplained. A subgroup of patients with the myeloproliferative variant carry the new gene rearrangement FIP1L1-PDGFRA, which produces a constitutively active tyrosine kinase often responsive to antityrosine kinase therapy with imatinib mesylate. A newly developed drug currently being tested in clinical trials is the humanized monoclonal antibody against human interleukin-5. Interleukin-5 positively regulates eosinophil growth, activation, survival and tissue recruitment. As such, anti-interleukin-5 therapy is expected to be very useful, especially for patients with the clonal T-cell variant that secretes high levels of interleukin-5. This review will analyze the etiology, classification and new options for the treatment of the hypereosinophilic syndromes, with particular emphasis on anti-interleukin-5 therapy.

Anti‐IL‐5 recombinant humanized monoclonal antibody (Mepolizumab) for the treatment of atopic dermatitis

Eosinophils may play an important role in the pathogenesis of atopic dermatitis (AD). Interleukin-5 is essential for eosinophil growth, differentiation and migration. A monoclonal antibody to human interleukin-5 (mepolizumab) was developed for atopic diseases. This study was designed to study the effect of mepolizumab in AD. Two single doses of 750 mg mepolizumab, given 1 week apart, were studied in patients with moderate to severe AD using a randomized, placebo-controlled parallel group design. The primary endpoint of 'success' to treatment was defined as the percentage of patients with at least 'marked improvement' after 2 weeks as assessed by the Physician's Global Assessment of Improvement (PGA). Furthermore, SCORing AD (SCORAD), pruritus scoring, number of blood eosinophils and serum thymus and activation-regulated chemokine (TARC) values served as secondary endpoints. Fluticasone propionate cream 0.05%, once daily could be used as rescue medication from day 16 if no improvement was recorded. Eighteen patients received mepolizumab and 22 placebo treatment. Peripheral blood eosinophil numbers were significantly reduced in the treatment group compared with placebo (P < 0.05). No clinical success was reached by PGA assessment (P = 0.115), SCORAD (P = 0.293), pruritus scoring and TARC values in the mepolizumab-treated group compared with placebo. However, modest improvement (<50% improvement) assessed by PGA was scored significantly more in the mepolizumab-treated group compared with placebo (P < 0.05). Two single doses of 750 mg mepolizumab did not result in clinical success in patients with AD, despite a significant decrease in peripheral blood eosinophils.

Hypereosinophilia in an Islet Transplant Recipient

A 38-year-old male subject, with a 36-year history of type 1 diabetes mellitus, received two allogeneic islet infusions with a steroid-free immunosuppressive therapy (1) resulting in insulin independence for 15 months and partial graft function for the following 7 months. Ten months posttransplant, progressive elevation of eosinophil count occurred reaching a peak value of 6,350 cells/mm3. Clinically he complained of scalp and upper torso itching exacerbated by exercise and hot water (treated with antihistamines) associated with intermittent rash. A skin biopsy demonstrated eosinophilic infiltrate, IgE antibody tests (for allergies) were negative, and thyroid function was normal. There was no weigh loss, upper and lower respiratory tract symtomatology, abdominal pain, or dyspepsia. After commencing immunosuppression, the patient suffered intermittent diarrhea, severe at times. Stool samples were consistently negative for infectious organism except on one occasion when giardia and iodamoeba were detected, successfully treated with metronidazole. A GI biopsy was normal. The diarrhea was presumed secondary to sirolimus. Causes for reactive eosinophilia were sought; infections, parasitic (angiostrongyliasis, capillariasis, fascioliasis, trichinosis), viral (HIV, HTLV, CMV, Parvovirus), and fungal (aspergillosis, candidiasis) were excluded. Malignancy was sought, chest and abdomen CT showed no masses or splenomegaly. Connective tissue diseases were excluded. An echocardiogram was normal. Drug reaction was evaluated by transiently removing all medications other than immunosuppression, no effect on eosinophilia was noted. A bone marrow biopsy demonstrated normocellularity with mild eosinophilia, without immunophenotypic abnormalities or clonality in any cell population. Interleukin-5 levels (an eosinophil growth factor) and serum tryptase (sign of mast cell degranulation) were slightly elevated. In our patient the most likely diagnosis was idiopathic hypereosinophilic syndrome (HES) associated with mast cell activation. Immunosuppressive drugs were discontinued, without alteration in eosinophil levels excluding sensitivity to the medications. The graft was at this point rejected (Fig. 1). Progression of eosinophilia argued against hypereosinophilia secondary to graft rejection.FIGURE 1. White blood cell and eosinophil counts during the clinical follow-up of the patient after transplantation. Important events in the clinical history of the patient is highlighted in the with arrows and relative legends.Eosinophilias can be classified into reactive forms, e.g., secondary to atopy or parasitic infestation, malignancies including lymphomas, connective tissue diseases, GVHD, bone marrow transplantation (2), and graft rejection, and nonreactive forms, e.g., chronic eosinophilic leukemia (CEL), systemic mastocytosis (SM) with eosinophilia, T-cell clonal and non-clonal disease-associated eosinophilia, and HES (3,4). HES is a collective term for those that cannot otherwise be classified, and, while the risk of malignancy is unclear, there is a risk of end-organ damage from the serum eosinophil granule proteins. The recent discovery of cytogenetic abnormality (fusion of the FIP1L1 gene with the platelet-derived growth factor receptor alpha gene) and therapeutic sensitivity to the tyrosine kinase inhibitor Imatinib in some forms of HES is leading to novel therapeutic approaches (5). Treatment with Imatinib mesylate (Gleevec 100 mg increased to 200 mg/day) was initiated and led to rapid remission of clinical signs and near normalization of laboratory abnormalities. Treatment will be continued indefinitely. Chronic (>6 months) hypereosinophilia, skin involvement, and therapeutic response to Imatinib are compatible with a diagnosis of primary HES. Since it developed during immunosuppressive therapy with tacrolimus, sirolimus, and daclizumab, a causal relationship cannot be excluded. Raffaella Poggioli Tatiana Froud David A. Baidal Jacqueline V. Ferreira Muhammad M. Hafiz Camillo Ricordi Diabetes Research Institute and Department of Surgery, University of Miami School of Medicine, Miami, Florida Gerald J. Gleich Department of Dermatology, University of Utah Health Sciences Center, Salt Lake City, Utah Rodolfo Alejandro Diabetes Research Institute and Department of Medicine, University of Miami School of Medicine, Miami, Florida

Human interleukin-5 expression is synergistically regulated by histone acetyltransferase CBP/p300 and transcription factors C/EBP, NF-AT and AP-1

Interleukin-5 (IL-5) plays a central role in the growth and differentiation of eosinophils and contributes to several disease states including asthma. There has been considerable interest in the identification of the transcriptional mechanisms controlling the synthesis of this cytokine. The regulation of IL-5 message is primarily at the level of transcription and is likely to be controlled, to a large extent, by regulatory elements in the promoter region that can influence the transcriptional activity of the gene. In this study, we performed a series of transient transfection experiments with IL-5 promoter–reporter gene construct and expression plasmid for E1A, an inhibitor of CBP/p300, or for the CBP/p300-binding defective E1AΔ2-36. The results showed that E1A repressed IL-5 promoter activity, while E1AΔ2-36 had no effect on it. This suggested that CBP/p300 was involved in regulation of IL-5 gene expression. Transcriptional coactivator CBP/p300 and transcription factors C/EBP, NF-AT, and c-Fos synergistically activated IL-5 promoter. Furthermore, we found that ectopic expression of p300 increased endogenous IL-5 mRNA expression. Thus, the histone acetyltransferase (HAT) activity of CBP/p300 was required to activate IL-5 expression. This report provided evidence, for the first time, that CBP/p300 was involved in IL-5 gene expression and the HAT activity was important in regulation of IL-5 expression.

CpG Oligodeoxynucleotides: A Novel Therapeutic Approach For Atopic Disorders

Atopic disorders such as allergic rhinitis, asthma and atopic dermatitis are associated with skewing of immune responses towards a TH2 phenotype, resulting in eosinophilic inflammation. TH2 cytokines promote eosinophil growth, migration and activation, mast cell differentiation, and IgE production, and are candidate mediators of pathologic abnormalities in asthma and other atopic diseases. There has been a significant increase in the prevalence of allergic disorders over the past several decades. Recent epidemiological studies suggest that reduced early-life exposure to strong TH1 stimuli in industrialized counties has skewed the TH1/TH2 balance towards TH2 responses. Improved hygiene, vaccination, and use of antibiotics may contribute to this imbalance. In the last half of the twentieth century we have seen the use of multiple agents to treat atopic disorders, ranging from antihistamines, steroids and leukotriene modifiers to anti-IgE antibodies. All these agents can block symptoms but do not significantly modify the course of the disease. Recent attempts to restore TH1/TH2 balance by blocking TH2 cytokines or inducing TH1 cytokines, have not only failed to alter the outcome of atopic diseases but, in some cases, have caused significant adverse effects. An alternate method of suppressing TH2 responses takes advantage of the innate immune response to bacterial DNA. Oligodeoxynucleotides (ODN) containing sequence motifs centered on unmethylated CG dinucleotides (CpG ODN) resemble bacterial DNA, and like bacterial DNA are immunostimulatory; we and others have shown that CpG ODN can suppress TH2-mediated atopic inflammation without requiring the induction of TH1-type cytokines. These agents may represent a novel therapeutic approach toward restoring immune tolerance in atopic individuals.

Lyn tyrosine kinase is important for IL-5-stimulated eosinophil differentiation.

IL-5 plays a pivotal role in growth and differentiation of eosinophils. The signal transduction mechanism of IL-5Ralpha is largely unknown. We have demonstrated that IL-5 induces tyrosine phosphorylation of IL-5Ralpha in eosinophils. To identify IL-5Ralpha-associated tyrosine kinases, we have examined the expression of Src family tyrosine kinases in eosinophils. Among the Src family members, Lyn, Hck, Fgr, and Lck are present in eosinophils, and, among these four kinases, only Lyn is associated with the IL-5Ralpha under basal conditions. We also confirm the association of Janus kinase (Jak)2 with IL-5Ralpha. Lyn kinase phosphorylates both IL-5Ralpha and betacR in vitro. The importance of Lyn kinase for eosinophil differentiation was studied using antisense oligodeoxynucleotides. Lyn antisense oligodeoxynucleotide blocks eosinophil differentiation from stem cells in a dose-dependent manner. The Jak2 inhibitor tyrphostin AG490 also inhibits eosinophil differentiation. The importance of Lyn for eosinophil differentiation was further studied using Lyn knockout mice. The IL-5-stimulated eosinophil differentiation from bone marrow cells is significantly inhibited in Lyn(-/-) mice as compared with that in control mice. We conclude that both Lyn and Jak2 play an essential role in IL-5Ralpha signaling, leading to eosinophil differentiation. The effect of Lyn appears to be relatively specific for the eosinophilic lineage.

Is asthma really due to a polarized T cell response toward a helper T cell type 2 phenotype?

Our understanding of pathogenesis has changed dramatically with time. Records, which date back to 1500 BC, indicate that was considered to be a disease caused by spirits. This viewpoint remained until 1678, when Thomas Willis described to be due to of bronchi by thick humors, swelling of their walls and obstruction from without. That was due to spasm of bronchial smooth muscles was first suggested by Sir John Floyer in 1698. With the advent of fiberoptic bronchoscopy in the 1970s and the use of modern molecular biology tools, it became clear that was a chronic inflammatory disorder of the airways mediated by a multitude of cell types and inflammatory mediators. Mast cells and eosinophils were initially believed to play a central role in driving the airway inflammation associated with asthma; however, the emphasis has now shifted to T lymphocytes. In particular, helper T type 2 (Th2) cells (a subset of T cells) are believed to play a central role in initiating and orchestrating the asthmatic airway inflammatory response (1). The hypothesis for asthma was first suggested by Mosmann in 1989 (2), who had earlier discovered the presence of two distinct subtypes of helper T cells in mice, namely, Th1 and Th2 (3). These two subclasses of helper T cells differ in their production of cytokines and are reciprocally inhibitory. The Th2 lymphocytes produce interleukin 4 (IL-4), IL-5, IL-9, and IL-13, which activate mechanisms important in defense against parasites and in allergic inflammation. Such mechanisms include IgE production, mast cell differentiation, and eosinophil growth, migration, and activation. Th1 lymphocytes produce interferon gamma (IFN-gamma ) and IL-2, which activate mechanisms important in defense against viruses and bacteria (2). A mechanism accounting for the predominance of Th2 cells in asthmatic airway was suggested by the discovery that the context in which antigen-presenting cells process and present allergens critically influences the pattern of helper T cell differentiation. The Th2 hypothesis for describes that is caused by a relative increase in Th2 cellular response in combination with a decrease in Th1 (helper T type 1) response. The consequent alteration in cytokine milieu (most likely in the lung), with excess Th2 products (e.g., IL-4, IL-5, and IL-13) in concert with decreased Th1 products (e.g., IFN-gamma and IL-12), is predicted to drive the phenotype (4). Evidence of such a shift in the Th1/Th2 balance derives from studies of in cellular and murine models, where Th cell polarization and allergen dependence of Th2 responses are most easily defined and from human studies that profile cytokine production. Although this viewpoint has remained popular over the last decade, more recent studies suggest that the Th2 hypothesis for is too simplistic (5). We now provide evidence to argue against the Th2 hypothesis for asthma.

Molecular and cellular mechanisms of allergic disease

The molecular and cellular mechanisms mediating the allergic inflammatory cascade involve multiple mediators, cell types, and pathways. Of particular interest are the pathways regulated by the T H2 lymphocyte, which result in release of IL-4 (important to IgE synthesis) and IL-5 (important to eosinophil proliferation). IL-4 regulates differentiation of naïve T H0 cells to develop a T H2 phenotype and stimulates B cells to produce IgE. Cross-linking by allergen of IgE affixed to high-affinity receptors on mast cells and basophils triggers degranulation and the release of preformed inflammatory mediators (important to the early phase response), and subsequently initiates synthesis and the release of lipid mediators and cytokines (which may contribute to the late phase response). Eosinophils may also play a prominent role in the development of bronchial hyperreactivity. IL-5, which is a lineage-specific eosinophil growth factor, increases the formation of eosinophils from progenitor cells and, in concert with CCR3 active chemokines, increases their trafficking to sites of allergic inflammation. An improved understanding of the basic mechanisms of allergic inflammation has led to the discovery of molecular targets involved in the initial events of the inflammatory cascade. Potential targets for the development of novel therapies for allergic disease include IgE, the T H2 lymphocyte, and T H2–derived cytokines, IL-4 and IL-5. (J Allergy Clin Immunol 2001;108:S65-71.)

Targeting cytokines in asthma therapy: round one

Eosinophils have long been linked with asthma, but only recently have striking advances been made in the understanding of the mechanisms of eosinophil accumulation in the airways. One key advance was recognition that airway eosinophilia is orchestrated by the actions of airway CD4+ T cells. In patients with asthma, these cells are mainly of the Th2 subclass, whereas normally the Th1 subclass predominates. 1 Robinson DS Hamid Q Ying S et al. Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma. N Engl J Med. 1992; 326: 298-304 Crossref PubMed Scopus (2551) Google Scholar These two subclasses of T-helper cells differ in their production of cytokines and are reciprocally inhibitory. Th2 lymphocytes produce interleukins 4, 5, 9, and 13, which activate mechanisms important in defence against parasites and in allergic inflammation. Such mechanisms include IgE production, mast-cell differentiation, and eosinophil growth, migration, and activation. Th1 lymphocytes produce interferon-γ and interleukin-2, which activate mechanisms important in defence against viruses and bacteria. 2 Mosmann TR Coffman RL TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol. 1989; 7: 145-173 Crossref PubMed Scopus (6816) Google Scholar Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyper-responsìveness, and the late asthmatic responseA single dose of monoclonal antibody to IL-5 decreased blood eosinophils for up to 16 weeks and sputum eosinophils at 4 weeks, which has considerable therapeutic potential for asthma and allergy. However, our findings question the role of eosinophils in mediating the late asthmatic response and causing airway hyper-responsiveness. Full-Text PDF Effects of recombinant human interleukin-12 on eosinophils, airway hyper-responsiveness, and the late asthmatic responseWe have shown that IL-12 lowers numbers of blood and sputum eosinophils, but without any significant effects on airway hyper-responsiveness or the late asthmatic reaction. This questions the role of eosinophils in mediating these reactions, and has important implications for development of new anti-inflammatory treatments. Full-Text PDF

Interleukin 5 and granulocyte-macrophage colony-stimulating factor levels in bronchoalveolar lavage fluid in interstitial lung disease.

The purpose of this study was to evaluate the role of several eosinophil growth factors including interleukin (IL)-5, interleukin (IL)-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the pathogenesis of interstitial lung disease with eosinophilia. IL-5, IL-3 and GM-CSF in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay (ELISA) in patients with eosinophilic pneumonia (EP), bronchiolitis obliterans organizing pneumonia (BOOP), idiopathic pulmonary fibrosis (IPF), sarcoidosis and healthy volunteers. IL-5 in BALF was high only in patients with EP. IL-3 in BALF was undetectable in the majority of patients with these diseases. GM-CSF in BALF was detectable in 30-67% of each group of patients. In patients with BOOP and IPF, the number of eosinophils in BALF was higher in patients with detectable GM-CSF than in patients in whom GM-CSF was below the detection limit. Eosinophil cationic protein (ECP) was detected in all patients with EP and some with BOOP and IPF. There was a significant correlation between ECP levels and percentage or number of eosinophils in BALF. The results suggest the possibility that interleukin 5 in eosinophilic pneumonia, and granulocyte-macrophage colony-stimulating factor in bronchiolitis obliterans organizing pneumonia and idiopathic pulmonary fibrosis may play important roles in eosinophil recruitment in the lung. Activation of eosinophils in the lung is likely to be induced by both interleukin 5 and granulocyte-macrophage colony-stimulating factor.

738 Abnormal clones of T cells producing interleukin-5 in idiopathic eosinophilia

Background The cause of persistent eosinophilia and the hypereosinophilic syndrome is unknown. Recent work suggests that in some patients with the hypereosinophilic syndrome, a clone of abnormal T cells produces large amounts of interleukin-5, a cytokine required for the growth and differentiation of eosinophils. We examined T-cell surface markers, rearranged T-cell–receptor genes, and in vitro production of cytokines by T cells from patients with idiopathic eosinophilia. Methods The expression of surface molecules on T cells was measured by flow cytometry. Cytokine expression was measured by enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemical analysis. To identify dominant (clonal) rearrangements of the T-cell receptor within the lymphocyte population, Southern blot analysis (β chain) and the polymerase chain reaction (γ chain) were performed according to standard protocols. Results Among 60 patients with idiopathic eosinophilia, 16 had circulating T cells with an aberrant immunoph...

DNase I footprinting of the human interleukin-5 gene promoter.

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.

Overexpression of CD44 on alveolar eosinophils with high concentrations of soluble CD44 in bronchoalveolar lavage fluid in patients with eosinophilic pneumonia.

High levels of interleukin-5 (IL-5) are found in the alveolar space of patients with eosinophilic pneumonia (EP). IL-5 promotes the growth and differentiation of eosinophils, as well as activating these cells. IL-5 also induces the expression of CD44 on eosinophils in vitro. To evaluate the contribution of CD44 to the pathogenesis of EP, we examined the expression of CD44 on eosinophils in bronchoalveolar lavage fluid (BALF) and measured the concentration of soluble CD44 (sCD44) in BALF from patients with EP. The concentrations of IL-5, sCD44, and hyaluronic acid (HA) were measured in BALF. The expression levels of CD44 on eosinophils in BALF and peripheral blood in patients with EP were compared. The expression of CD44 on alveolar eosinophils and the concentration of sCD44 were increased in BALF of patients with EP. There was a significant correlation between IL-5 and sCD44 in BALF. A high concentration of HA was observed in BALF of EP patients. Our results suggest that the high expression of CD44 on eosinophils probably results from upregulation by IL-5 and could be important in the pathogenesis of EP.